Identification and Characterization of Mikcc-Type MADS-Box Genes In

International Journal of Molecular Sciences

Article Identiﬁcation and Characterization of MIKCc-Type MADS-Box Genes in the Flower Organs of Adonis amurensis

Lulu Ren †, Hongwei Sun †, Shengyue Dai, Shuang Feng, Kun Qiao, Jingang Wang, Shufang Gong * and Aimin Zhou *

College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin 150030, China; [email protected] (L.R.); [email protected] (H.S.); [email protected] (S.D.); [email protected] (S.F.); [email protected] (K.Q.); [email protected] (J.W.) * Correspondence: [email protected] (S.G.); [email protected] (A.Z.) † Authors contributed equally to the work.

Abstract: Adonis amurensis is a perennial herbaceous flower that blooms in early spring in northeast China, where the night temperature can drop to −15 ◦C. To understand flowering time regulation and floral organogenesis of A. amurensis, the MIKCc-type MADS (Mcm1/Agamous/ Deficiens/Srf)-box genes were identified and characterized from the transcriptomes of the flower organs. In this study, 43 non-redundant MADS-box genes (38 MIKCc, 3 MIKC*, and 2 Mα) were identified. Phylogenetic and conserved motif analysis divided the 38 MIKCc-type genes into three major classes: ABCDE model (including AP1/FUL, AP3/PI, AG, STK, and SEPs/AGL6), suppressor of overexpression of constans1 (SOC1), and short vegetative phase (SVP). qPCR analysis showed that the ABCDE

model genes were highly expressed mainly in flowers and differentially expressed in the different tissues of flower organs, suggesting that they may be involved in the flower organ identity of

Citation: Ren, L.; Sun, H.; Dai, S.; A. amurensis. Subcellular localization revealed that 17 full-length MADSs were mainly localized in Feng, S.; Qiao, K.; Wang, J.; Gong, S.; the nucleus: in Arabidopsis, the heterologous expression of three full-length SOC1-type genes caused Zhou, A. Identification and early flowering and altered the expression of endogenous flowering time genes. Our analyses provide c Characterization of MIKCc-Type an overall insight into MIKC genes in A. amurensis and their potential roles in floral organogenesis MADS-Box Genes in the Flower and flowering time regulation. Organs of Adonis amurensis. Int. J. Mol. Sci. 2021, 22, 9362. https:// Keywords: Adonis amurensis; MADS-box genes; ABCDE model; SOC1; flowering; flower organs doi.org/10.3390/ijms22179362

Academic Editor: Zsóﬁa Bánfalvi 1. Introduction Received: 24 July 2021 The MADS (Mcm1/Agamous/Deﬁciens/Srf)-box transcription factor gene family Accepted: 26 August 2021 Published: 28 August 2021 plays an important role in the regulation of plant growth and development [1]. This large gene family is divided into two types, types I and II, based on phylogenetic relationships

Publisher’s Note: MDPI stays neutral of the conserved MADS-box domain [2]. In plants, the type-I genes are further divided α β γ c with regard to jurisdictional claims in into M ,M , and M subfamilies, and the type-II genes into the MIKC -type and MIKC*- published maps and institutional affil- type [3,4]. The term MIKC originated from the four major domains, including MADS (M), iations. intervening (I), keratin-like (K), and C-terminal (C) [5]. The MIKCc-type MADS-box genes are involved in flowering time regulation and floral organ identity. For example, flowering locus C (FLC), suppressor of overexpression of constans1 (SOC1), and short vegetative phase (SVP) were reported to be key regulators of flowering time; the FLC gene encodes a specific MADS domain protein that acts as a Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. repressor of flowering, and SOC1 and SVP are important control factors of flowering time This article is an open access article in the vernalization and ambient temperature pathways, respectively [6–8]. Moreover, the distributed under the terms and MADS-box genes of the extended ABCDE model explain how the different floral organ c conditions of the Creative Commons identities belong to the MIKC subgroups [9], namely, sepals (A + E), petals (A + B + E), Attribution (CC BY) license (https:// stamens (B + C + E), carpels (C + E), and ovules (D + E). In this model, class A con- creativecommons.org/licenses/by/ tains APETALA1 (AP1) and FRUITFULL (FUL); class B contains PISTILLATA (PI) and 4.0/). APETALA3 (AP3); class C contains AGAMOUS (AG); class D contains SEEDSTICK (STK);

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and class E contains SEPALLATA genes (SEP1, SEP2, SEP3, and SEP4)[9]. The ABCDE model was initially established in Arabidopsis and also works for most eudicots. The identification and characterization of MADS-box genes are extremely important for the study of flowering time regulation and flower organ development in plant species. Currently, MADS-box genes were identified and characterized in various plant species with reference genomic resources, including Arabidopsis [5], rice [10], Zea mays [11], soybean [12], Raphanus sativus [13], Phyllostachys edulis [14], and Jatropha curcas [15]. However, gene identification in non-model plant species without genomic resources is difficult. Recently, multiple functional MADS-box genes were identified and characterized in Lilium formosanum and Rosa chinensis using transcriptome sequencing [16,17], suggesting the feasibility of this method. A. amurensis is a perennial herbaceous flower in the family Ranunculaceae, which is naturally distributed in northeast China. A. amurensis can blossom before the ice and snow melts in the early spring, when the temperature is about −15 ◦C (night) and 10 ◦C (day) [18]. Therefore, it is the ideal plant species to study flowering control at extreme low temperatures. In this study, 43 non-redundant MADS-type transcripts were extensively identified from transcriptomes of flower organs at multiple development stages. Further, the conserved motifs, expression patterns, and subcellular localization of the expressed proteins were investigated. Moreover, the function of three SOC1-type MADS was characterized by heterogenous expression in Arabidopsis. This study will serve as a useful reference for further functional analyses of candidate genes involved in the flowering time control and flower development of A. amurensis at low temperatures.

2. Results 2.1. Identification and Annotation of MADS-Box Genes in A. amurensis In our previous study, the 3216 transcription factors (TFs) in the A. amurensis flower organs at six developmental stages were identified and classified by transcriptome sequencing. Of these TFs, 91 MADS-type transcripts were annotated [18]. After removing the redundants, 43 MADS-box putative genes were finally obtained (Table S1). The phylogenetic tree and conserved motifs of these 43 AaMADS putative genes were constructed and identified (Figure1A,B). Among the corresponding proteins, motifs 1 and 2 were identified and were conserved MADS domains. Motifs 3 and 5, which were keratin (K) domains, were identified in 27 AaMADS proteins (Figure1B). The 43 AaMADS putative proteins were named and classified according to the phylogenetic relationship between AaMADS and Arabidopsis MADS (also known as the Agamous-like, AGL) proteins. They were subdivided into 3 major classes, Mα, MIKC*, and MIKCc, of which MIKCc was divided into 7 sub- classes, including SVP (four members), A-class (AP1 and FUL, eight members), B-class (PI and AP3, eight members), C-class (AG, one member), D-class (STK, one member), E-class (SEP1/2/3 and AGL6, eight members), and SOC1 (four members) (Figure1C ). Interest- ingly, only two MIKC*-type AtAGL65 homologous proteins (CL19409.C1 and CL10680.C2) shared motifs 15, 16, 17, and 19 (Figure1C).

(Figure4A). In the class A genes, ﬁve AP1 ( U114330, U119430, U74157, CL3032.C1, and CL3350.C5) were expressed slightly higher in the petals, sepals, and stamens, while one FUL (CL26633.C2) was expressed higher in the petals (Figure4B). In the class B genes, ﬁve AP3 (CL10616.C1, CL27078.C1, U1341, U31032, and U123412) and two PI (CL7220.C2 and U23673) were highly expressed in the petals and stamens, while one PI (CL7507.C2) was highly expressed in the sepals and stamens (Figure4C). One C-class gene AG ( CL18186.C3) and one D-class gene STK (U152236) were highly expressed in the stamens (Figure4D). In the class E genes, one SEP1 (CL27155.C2) was highly expressed in the stamens, while Int. J. Mol. Sci. 2021, 22, 9362 3 of 12 one SEP1 (CL20429.C1) and two AGL6 (U113876 and U7086) were highly expressed in the petals, sepals, and stamens (Figure4D).

FigureFigure 1. Classification 1. Classification and and conserved conserved motifs motifs ofof MADS-boxMADS-box putative putative proteins proteins in Adonis in Adonis amurensis. amurensis.(A) The ( phylogeneticA) The phylogenetic tree tree ofof the the identified identified 43 AaMADS putativeputative proteins proteins and and their their conserved conserved motifs. motifs. (B) ( MotifsB) Motifs 1 to 1 20 to are 20 indicated are indicated by different by different coloredcolored boxes. boxes. The Thecombined combined probability probability values values are are shown shown on thethe leftleft side. side. (C ()C Classification) Classification and and naming naming of 43 of AaMADS 43 AaMADS putativeputative proteins proteins based based on on the the similarity similarity to ArabidopsisArabidopsisMADS-box MADS-box homologous homologous proteins. proteins. SVP: Short SVP: vegetative Short vegetative phase; AG: phase; AG: Agamous;Agamous; AGL: AGL: Agamous-like; Agamous-like; TT16: TT16: TRANSPARENT TRANSPARENT TESTA 16;TESTA PI: PISTILLATA; 16; PI: PISTILLATA; AP: APETALA; AP: STK: APETALA; SEEDSTICK; STK: FUL: SEED- STICK;FRUITFULL; FUL: FRUITFULL; SEP: SEPALLATA; SEP: SEPALLATA; SOC1: Suppressor SOC1: of Suppresso overexpressionr of overexpression of CONSTANS1; of MIKC: CONSTANS1; MADS (M), MIKC: intervening MADS (I), (M), interveningkeratin-like (I), keratin-like (K), and C-terminal (K), and (C) C-terminal domain. (C) domain.

2.2. Expression of AaMADS Genes in the Flowers of A. amurensis Based on transcriptome data, the expression of the 43 AaMADS genes showed two different patterns in the A. amurensis flower organs at six developmental stages. One is the high expression of AaMADS genes in five development stages [young alabastrum (YA), visible color alabastrum (VCA), full bloom stage (FBS), and senescing flower stage (SFS)], while the other is the high expression of AaMADS genes only in the flower bud differentiation (FBD) stage (Figure 2). To understand their expression patterns, the expression of 24 AaMADS genes classified in the ABCDE model in stems, leaves, flowers, and achenes was exam- ined. qPCR analysis showed that AaMADS genes belonging to the A-, B-, C-, D-, and E- classes were generally higher expressed in flowers than in stems, leaves, and achenes (Fig- ure 3). Further, their expression in the four tissues of the flower organs was investigated. A. amurensis flower includes about 7–9 sepals (pale grayish purple), about 10–13 petals (yellow), ellipsoid ovary, stigma unsmooth, sac-like anther, and spherical pollen grains (Figure 4A). In the class A genes, five AP1 (U114330, U119430, U74157, CL3032.C1, and CL3350.C5) were expressed slightly higher in the petals, sepals, and stamens, while one FUL (CL26633.C2) was expressed higher in the petals (Figure 4B). In the class B genes, five AP3 (CL10616.C1, CL27078.C1, U1341, U31032, and U123412) and two PI (CL7220.C2 and U23673) were highly expressed in the petals and stamens, while one PI (CL7507.C2) was highly expressed in the sepals and stamens (Figure 4C). One C-class gene AG (CL18186.C3) and one D-class gene STK (U152236) were highly expressed in the stamens

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Int. J. Mol. Sci. 2021, 22, 9362 (Figure 4D). In the class E genes, one SEP1 (CL27155.C2) was highly expressed4 of 12 in the sta- Int. J. Mol. Sci. 2021, 22, 9362 mens, while one SEP1 (CL20429.C1) and two AGL6 (U113876 and U7086)4 were of 12 highly expressed in the petals, sepals, and stamens (Figure 4D). (Figure 4D). In the class E genes, one SEP1 (CL27155.C2) was highly expressed in the stamens, while one SEP1 (CL20429.C1) and two AGL6 (U113876 and U7086) were highly expressed in the petals, sepals, and stamens (Figure 4D).

Figure 2. Expression pattern clustering of 43 AaMADS putative genes in floral organs at six devel- Figure 2. 2.Expression Expression pattern pattern clustering clustering of 43 AaMADS of 43 AaMADSputative genesputative in floral genes organs in floral at sixdevelop- organs at six developmental stages (FBD: flower bud differentiation; YA: young alabastrum; VCA: visible color alabastrum;mentalopmental EFS: stages early stages (FBD: flowering (FBD: flower flower budstage; differentiation; FBS: bud full differentiation; bloom YA: st youngage; SFS: alabastrum;YA: senescing young VCA: alabastrum; flower visible stage). color VCA: The alabastrum; levels visible of color alabas- expressionEFS:trum; early EFS: floweringof earlyeach gene flowering stage; during FBS: fullFBD,stage; bloom YA, FBS: VCA, stage; full EFS, SFS: bloom senescingFBS, andstage; SFS flower SFS: are stage).indicated senescing The by levels flowerred/blue of expression stage). rectan- The levels of gles,ofexpression each where gene red duringof eachrectangles FBD, gene YA, representduring VCA, EFS, FBD, the FBS, upregulati YA, and VCA, SFSon are EFS, of indicated genes, FBS, bywhileand red/blue SFS blue are rectangles rectangles, indicated whererepresent by redred/blue rectan- downregulation.rectanglesgles, where represent red rectangles the upregulation represent of genes, the while upregulati blue rectangleson of represent genes, downregulation.while blue rectangles represent downregulation.

Figure 3. Expression analysis of 24 AaMADS genes in different tissues, including stems, leaves, flowers, Figure 3. Expression analysis of 24 AaMADS genes in different tissues, including stems, leaves, and achene by qPCR. The AaActin gene was used as an internal control, and the transcript level in stems flowers, and achene by qPCR. The AaActin gene was used as an internal control, and the transcript was set as 1.0. Asterisks indicate significant differences in gene expression levels between other tissues and the stem (* p < 0.05; ** p < 0.01; Student’s t-test). Error bars represent the SE (n = 3).

Figure 3. Expression analysis of 24 AaMADS genes in different tissues, including stems, leaves, flowers, and achene by qPCR. The AaActin gene was used as an internal control, and the transcript

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Int. J. Mol. Sci. 2021, 22, 9362 level in stems was set as 1.0. Asterisks indicate significant differences in gene expression5 of 12 levels between other tissues and the stem (* p < 0.05; ** p < 0.01; Student’s t-test). Error bars represent the SE (n = 3).

Figure 4.4.Organ-specific Organ-specific expression expression analysis analysis of 20 ofAaMADS 20 AaMADSgenes genes classified classified in the in ABCDE the ABCDE model model in thein the flower flower of of A.A. amurensis .(. A(A)) The The flower flower morphology morphology and itsand anatomy, its anatomy, including including petals, calyx, petals, calyx, stamens, and and pistils pistils (a1 (a1), simple), simple pistil pistil (a2) ( anda2) and stigma stigma (a4), simple(a4), simple anther anther (a3) and (a3 pollen) and grain pollen (a5 grain). (a5). qPCR analysisanalysis of of A-class A-class (B ),(B B-class), B-class (C), ( andC), and C-, D-, C-, and D-, E-class and E-class (D) AaMADS (D) AaMADSgene expression gene expression in in different flower flower structures. structures. The TheAaActin AaActingene wasgene used was as anused internal as an control, internal and control, the transcript and the level transcript levelin petals in petals was set was as 1.0. set Asterisks as 1.0. Asterisks indicate significantindicate significant differences differences in gene expression in gene levels expression between levels be- tweenother tissues other andtissues petals and (* ppetals< 0.05; (* ** pp << 0.05; 0.01; ** Student’s p < 0.01;t-test). Student’s Error t bars-test). represent Error bars the SE represent(n = 3). the SE (n = 3).

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2.3.2.3. Subcellular Subcellular Localization Localization of of AaMADS AaMADS Proteins Proteins TheThe subcellularsubcellular localizationlocalization of of 17 17 full-length full-length AaMADS AaMADS proteins proteins classified classified in the SOC1-,in the SOC1-,SVP-, TT16-type, SVP-, TT16- andtype, ABCDE and modelABCDE was model investigated was investigated by transient by expression transient expression with green withfluorescent green fluorescent protein (GFP) protein fused (GFP) with AaMADSfused with proteins AaMADS in tobacco proteins leaves. in tobacco Confocal leaves. ob- Confocalservations observations showed the showed fluorescent the fluorescen signals of allt signals 17 AaMADS-GFP, of all 17 AaMADS-GFP, including three including SOC1 three(CL8076.C1, SOC1 CL6237.C1,(CL8076.C1, and CL6237.C1, CL10716.C2), and twoCL10716.C2), SVP (CL28194.C1 two SVP and (CL28194.C1 CL28920.C2), and one CL28920.C2),TT16 (CL2264.C2), one TT16 two A-class (CL2264.C2), (U11162 two and U119430),A-class (U11162 four B-class and (CL7220.C2,U119430), four CL7507.C2, B-class (CL7220.C2,U31032, and CL7507.C2, CL10616.C1), U31032, one D-class and CL10616. (U152236),C1), and one four D-class E-class (U152236), (CL28019.C1, and U158374, four E- classCL20429.C1, (CL28019.C1, and U7086), U158374, were CL20429.C1, mainly localized and U7086), in the nucleus, were mainly which waslocalized stained in bythe DAPI nucleus,(Figure which5A–G). was stained by DAPI (Figure 5A–G).

Figure 5. Subcellular localization localization of of 17 17 AaMADS AaMADS fused fused with with green green fluorescent fluorescent protein protein (GFP) in(GFP) tobacco in leaves.tobacco The leaves. 17 AaMADS The 17 AaMADSbelonged tobelonged the SOC1 to the type SOC1 (A), type SVP ( typeA), SVP (B), type TT16 (B type), TT16 (C), type A-class (C), (A-classD), B-class (D), ( EB-class), D-class (E), (D-classF), and ( E-classF), and (E-classG) groups, (G) groups, respectively. GFP fluorescence is green and the nuclear dye DAPI is blue. Merge is created by merging the GFP, respectively. GFP fluorescence is green and the nuclear dye DAPI is blue. Merge is created by merging the GFP, DAPI, and DAPI, and bright-field images. Scale bar = 10 µm. bright-field images. Scale bar = 10 µm. 2.4. Characterization of Three SOC1-Type AaMADS Genes 2.4. Characterization of Three SOC1-Type AaMADS Genes SOC1 is a key flowering regulator, which was reported to be associated with the final SOC1 is a key flowering regulator, which was reported to be associated with the final steps of floral organ development [19]. Thus, the function of three full-length SOC1-type steps of floral organ development [19]. Thus, the function of three full-length SOC1-type AaMADSAaMADS ((CL8076.C1CL8076.C1,, CL6237.C1CL6237.C1,, and and CL10716.C2CL10716.C2)) was was investigated investigated by by heterologous heterologous ex- ex- pressionpression in in Arabidopsis driven by the CaMV 35S promoter. CL8076.C1, CL6237.C1, and

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CL10716.C2 had 66.4%, 53.1%, and 56.3% amino acid sequence identity with AtSOC1, respectivelyCL10716.C2 had (Figure 66.4%,6A), 53.1%, and wereand 56.3% thus namedamino acid AaSOC1a, sequence AaSOC1b, identity with and AtSOC1, AaSOC1c, re- re- spectively (Figure 6A), and were thus named AaSOC1a, AaSOC1b, and AaSOC1c, respec- spectively. The transient expression of the plasmid of GFP-fused AaSOC1a/b/c showed tively. The transient expression of the plasmid of GFP-fused AaSOC1a/b/c showed that that they were localized into the nucleus in tobacco leaves (Figure5A). These constructs they were localized into the nucleus in tobacco leaves (Figure 5A). These constructs were were further transfected into Arabidopsis. Transgenic Arabidopsis lines overexpressing further transfected into Arabidopsis. Transgenic Arabidopsis lines overexpressing AaSOC1a-GFP, AaSOC1b-GFP, and AaSOC1c-GFP were identified by reverse transcription AaSOC1a-GFP, AaSOC1b-GFP, and AaSOC1c-GFP were identified by reverse transcrip- (RT) PCR (Figure6B and Figure S1). Phenotypic observations showed that transgenic tion (RT) PCR (Figure 6B and Figure S1). Phenotypic observations showed that transgenic plants (30.5 ± 0.7 days) overexpressing AaSOC1a-GFP, AaSOC1b-GFP, and AaSOC1c-GFP plants (30.5 ± 0.7 days) overexpressing AaSOC1a-GFP, AaSOC1b-GFP, and AaSOC1c-GFP ± floweredflowered earlierearlier than the the wild-type wild-type (WT) (WT) control control (40.3 (40.3 ± 1.51.5 days) days) (Figure (Figure 6C).6C). Further- Further- more,more, thethe expressionexpression of endogenous endogenous flowering flowering time time genes, genes, AtFLCAtFLC, AtFT, AtFT, and, and AtSOC1AtSOC1, in , in transgenictransgenic ArabidopsisArabidopsis andand WTWT waswas comparedcompared by qPCR,qPCR, and results showed that expres- expression ofsionAtFLC of AtFLCand andAtSOC1 AtSOC1was was significantly significantly lower lower inin all transgenic transgenic ArabidopsisArabidopsis thanthan in the in the WTWT (Figure(Figure7 7A–C).A–C). TheThe AtFT expressionexpression was was likewise likewise significantly significantly lower lower in transgenic in transgenic ArabidopsisArabidopsis overexpressing AaSOC1a-GFP AaSOC1a-GFP and and AaSOC1b-GFP AaSOC1b-GFP than than in the in theWT, WT, while while it it waswas higherhigher thanthan thethe WTWT inin transgenictransgenic ArabidopsisArabidopsis overexpressing AaSOC1c-GFP AaSOC1c-GFP (Figure (Figure 7). These7). These results results suggest suggest that that the the overexpression overexpression of of AaSOC1a/b/c AaSOC1a/b/c perturbatesperturbates the expressionexpres- ofsion endogenous of endogenous flowering flowering time time genes genes in Arabidopsis in Arabidopsis. .

FigureFigure 6.6. PhenotypicPhenotypic analysis of of transgenic transgenic ArabidopsisArabidopsis overexpressingoverexpressing three three SOC1-type SOC1-type AaMADSAaMADS genes.genes. ((AA)) AminoAmino acid sequence sequence alignment alignment of of thr threeee SOC1-type SOC1-type AaMADS AaMADS (CL8076.C1, (CL8076.C1, CL6237.C1, CL6237.C1, and CL10716.C2) with Arabidopsis AtSOC1 (At2g45660) protein. Semi-quantitative PCR detection and CL10716.C2) with Arabidopsis AtSOC1 (At2g45660) protein. Semi-quantitative PCR detection (B) and ﬂowering time phenotypes (C) of transgenic lines overexpressing three SOC1-type AaMADS genes. The AaActin gene was used as an internal control. WT: Wild-type.

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Int. J. Mol. Sci. 2021, 22, 9362 (B) and flowering time phenotypes (C) of transgenic lines overexpressing8 ofthree 12 SOC1-type AaMADS genes. The AaActin gene was used as an internal control. WT: Wild-type.

Figure 7. Relative expression level of endogenous flowering time genes in wild-type (WT) and transgenicFigure 7.Arabidopsis Relative overexpressingexpression levelAaSOC1a of endogenous-(A), AaSOC1b-( flowB), anderingAaSOC1c-GFP time genes(C) in genes. wild-type (WT) and trans- ThegenicAtActin Arabidopsisgene wasused overexpressing as an internal control, AaSOC1a and the- transcript(A), AaSOC1b level in WT- ( wasB), setand as 1.0.AaSOC1c-GFP Error (C) genes. The barsAtActin represent gene the was SE (n =used 3). Asterisks as an indicateinternal significant control, differences and the between transcript transgenic level lines in and WT was set as 1.0. Error WTbars plants represent (* p < 0.05; the ** pSE< 0.01;(n = Student’s 3). Asteriskst-test). FLC:indicate Flowering significant locus C; FT: di Floweringfferences locus between T. transgenic lines and 3.WT Discussion plants (* p < 0.05; ** p < 0.01; Student’s t-test). FLC: Flowering locus C; FT: Flowering locus T. The number of the MADS-box genes identified in various plant species shows great difference.3. Discussion For example, in Arabidopsis and rice, 107 and 75 MADS-box genes were annotated, respectively, while 42 and 39 MADS-box genes were identified in Phyllostachys edulis and DianthusThe number caryophyllus of ,the respectively, MADS-box using agenes genome-wide identified search in [5 various,10,14,19]. plant Using species shows great transcriptionaldifference. For sequencing, example, 58 MADS-boxin Arabidopsis genes wereand identifiedrice, 107 in and the flower75 MADS-box buds of genes were anno- R.tated, chinensis respectively,[17]. Similarly, while in our 42 study, and 4339 MADS-box MADS-box genes genes were identifiedwere identified from the in Phyllostachys edu- transcriptional data of A. amurensis flower organs using transcriptional sequencing. Both A.lis amurensis and Dianthusand Aquilegia caryophyllus coerulea belong, respectively, to the family Ranunculaceae.using a genome-wide In A. coerulea searchfrom [5,10,14,19]. Using Ranunculaceae,transcriptional 47 MADS-boxsequencing, genes 58 were MADS-box annotated usinggenes a genome-widewere identified search in [20 ].the flower buds of R. Thechinensis MIKC-type [17]. members Similarly, are the in most our common study, in 43 the MADS-box MADS-box gene genes family. were Among identified the from the tran- 107 MADS-box genes in Arabidopsis, 39 are MIKC-type [5], whereas among the 75 genes in rice,scriptional 38 are MIKC-type data of [10 A.]. Meanwhile,amurensis the flower identified organs 43 AaMADS usinggenes transcriptional in A. amurensis sequencing. Both A. containamurensis 38 MIKC andc-type Aquilegia and 3 MIKC*-type coerulea belong (Figure1 ).to The the vast family majority Ranunculaceae. of the identified In A. coerulea from Ranunculaceae, 47 MADS-box genes were annotated using a genome-wide search [20]. The MIKC-type members are the most common in the MADS-box gene family. Among the 107 MADS-box genes in Arabidopsis, 39 are MIKC-type [5], whereas among the 75 genes in rice, 38 are MIKC-type [10]. Meanwhile, the identified 43 AaMADS genes in A. amurensis contain 38 MIKCc-type and 3 MIKC*-type (Figure 1). The vast majority of the identified AaMADS are MIKCc-type members, which may be related with identifying genes from the flower organs of A. amurensis, as MIKCc is primarily involved in flowering time regulation and flower organ identity [6–9]. The 38 MIKCc genes contain 26 ABCDE model genes and 4 SOC1-type genes. qPCR analysis showed that 24 AaMADS genes belonging to the ABCDE model were expressed relatively high in the flowers (Figure 3),

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Int. J. Mol. Sci. 2021, 22, 9362 AaMADS are MIKCc-type members, which may be related with identifying genes from the 9 of 12 flower organs of A. amurensis, as MIKCc is primarily involved in flowering time regulation and flower organ identity [6–9]. The 38 MIKCc genes contain 26 ABCDE model genes and 4 SOC1-type genes. qPCR analysis showed that 24 AaMADS genes belonging to the whileABCDE in modelthe petals, were the expressed expression relatively of A- high (AP1 in theand flowers FUL), (FigureB- (AP33), and while PI in), and the petals, E- (SEP1 and AGL6the expression) class genes of A- was (AP1 slightlyand FUL higher.), B- (AP3 Classand API (),AP1 and) E-and (SEP1 E (AGL6and AGL6) genes) class were genes expressed slightlywas slightly higher higher. in the Class sepals. A (AP1 In the) and stamens, E (AGL6 class) genes B were(AP3 expressedand PI) and slightly class higher E (SEP1 in ) genes showedthe sepals. high In expression, the stamens, while class Bthe (AP3 classand C (PIAG) and) and class D ( ESTK (SEP1) genes) genes were showed highly high expressed inexpression, the pistils while (Figure the class 4). CGene (AG )expression and D (STK )of genes A-, wereB-, C-, highly D-, expressedand E-class in thein pistilsA. amurensis showed(Figure4 ).similarities Gene expression and differences of A-, B-, C-, found D-, and in E-class Arabidopsis in A. amurensis and othershowed species. similarities These results Arabidopsis suggestand differences that the foundABCDE in model genesand may other be species. involved These in the results flower suggest organ that identity the of A. ABCDE model genes may be involved in the flower organ identity of A. amurensis. Thus, a amurensismodel of.gene Thus, expression a model of patterns gene expression in A. amurensis patternsis proposed in A. amurensis (Figure8). is proposed (Figure 8).

FigureFigure 8. Hypothetical model model of of expression expression pattern pattern of AaMADS of AaMADSgenes. genes. Five colors Five representcolors represent five five classclass AaMADSAaMADS genes, genes, including including class class A, B,A, C,B,D, C, and D, and E. E. Some AaMADS sequences lack a full-length CDS due to the limitations of transcrip- Some AaMADS sequences lack a full-length CDS due to the limitations of transcriptome sequencing. We successfully cloned 17 full-length AaMADS and observed their tomesubcellular sequencing. expression We localization.successfully Transient cloned expression17 full-length in the AaMADS tobacco leaves and observed showed that their sub- cellularthe 17 AaMADS-GFP expression localization. were all localized Transient mainly ex inpression the nucleus, in suggestingthe tobacco their leaves function showed as that theTFs 17 (Figure AaMADS-GFP5). The 17 full-length were all localizedAaMADS genesmainly contain in the 3 nucleus, SOC1-type suggestingAaMADS, namedtheir function asAaSOC1a TFs (Figure, AaSOC1b 5)., andTheAaSOC1c 17 full-length. SOC1 isAaMADS a key transcription genes contain factor that 3 regulatesSOC1-type flower- AaMADS, nameding time AaSOC1a [21]. Heterologous, AaSOC1b expression, and AaSOC1c of AaSOC1a-GFP. SOC1 ,isAaSOC1b-GFP a key transcription, and AaSOC1c-GFP factor that regu- latesall caused flowering the early time flowering [21]. He ofterologousArabidopsis expression(Figure6). Similarly, of AaSOC1a-GFP heterologous, AaSOC1b-GFP expression , and AaSOC1c-GFPof SOC1 homologous all caused genes the from early various flowering plant of species Arabidopsis promotes (Figure the early 6). Similarly, flowering ofheterolo- Arabidopsis, for example, Z. mays [22], P. violascens [23], and Dendrobium nobile [24]. In gous expression of SOC1 homologous genes from various plant species promotes the early transgenic Arabidopsis overexpressing AaSOC1a-GFP, AaSOC1b-GFP, or AaSOC1c-GFP, floweringthe AtFLC ofgene Arabidopsis expression, for was example, significantly Z. mays suppressed [22], P. violascens compared [23], to the and WT Dendrobium (Figure7). nobile [24].AtFLC Inexpression transgenic inhibition Arabidopsis is a keyoverexpressing step in the flowering AaSOC1a-GFP of Arabidopsis, AaSOC1b-GFP[6]. The expression, or AaSOC1c- GFPof AtSOC1, the AtFLCin all gene transgenic expressionArabidopsis was significantlywas also significantly suppressed lower compared than the to wild the type.WT (Figure 7).Moreover, AtFLC expression the expression inhibition of flowering is alocus key Tstep (AtFT in )the was flowering also affected of relativeArabidopsis to the [6]. WT The expression(Figure7). of We AtSOC1 speculated in all that transgenicAaSOC1 Arabidopsismay functionally was also replace significantly endogenous lower flowering than the wild type. Moreover, the expression of flowering locus T (AtFT) was also affected relative to the WT (Figure 7). We speculated that AaSOC1 may functionally replace endogenous flowering time genes, such as AtSOC1 and AtFT, that further affect the flowering time of Arabidopsis. However, this hypothesis requires further study.

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time genes, such as AtSOC1 and AtFT, that further affect the ﬂowering time of Arabidopsis. However, this hypothesis requires further study.

4. Materials and Methods 4.1. Identification of MADS-Box Genes in A. amurensis In our previous study, the transcriptome of the floral organs of A. amurensis from six developmental stages, FBD, YA, VCA, EFS, FBS, and SFS, was assembled using Trinity [18]. All assembled unigenes were annotated by comparing the data available at the following public databases: NCBI non-redundant protein sequence (Nr), NCBI nucleotide sequence (Nt), Swiss-Prot protein, Kyoto Encyclopedia of Genes and Genomes (KEGG), euKaryotic Ortholog Groups (KOG), InterPro, and Gene Ontology (GO) databases, using BLAST2GO analysis with a cut-off E-value of 10−5. TFs in the annotated unigenes were predicted using the Plant Transcription Factor Database (PlantTFDB; available online: http://planttfdb.gao- lab.org/. TFs with the same annotated information and the longest unigene were selected. Finally, sequences of 43 MADS-box putative genes were obtained from the transcriptional datasets of A. amurensis floral organs. The transcriptome datasets were deposited in the NCBI Gene Expression Omnibus with accession number GSE126456.

4.2. Conserved Motifs and Phylogenetic Analysis of MADS-Box Proteins The conserved motifs of the 43 AaMADS putative proteins were identified by the multiple expectation for motif elicitation (MEME) tool (available online: https://meme- suite.org/meme/) according to the default parameters. Multiple sequence alignments were performed between MADS-box protein sequences from A. amurensis and Arabidopsis using the ClustalW software. The sequence of the Arabidopsis MADS protein family was taken from the TAIR website (available online: https://www.arabidopsis.org/). The phylogenetic tree was constructed by the neighbor-joining method using the molecular evolutionary genetics analysis (MEGA) 4.1 software (available online: http://www.megasoftware.net/). The amino acid sequences of the 43 putative AaMADS proteins are listed in Supplementary Data 1.

4.3. Expression-Pattern Clustering of AaMADS Genes The expression levels of 43 AaMADS putative genes in A. amurensis ﬂoral organs at six developmental stages (FBD, YA, VCA, EFS, FBS, and SFS) were calculated using the Fragments Per Kilobase of transcript per million mapped reads (FPKM) (Table S2).

4.4. Quantitative Real-Time PCR (qPCR) Analysis Total RNA from multiple organs (stems, leaves, ﬂowers, and achene) and tissues (calyx, petals, stamens, and pistils) of A. amurensis was extracted using the TRIzol reagent (9108, TaKaRa, Kusatsu, Japan) and reverse transcribed with PrimeScript RT reagent Kit with gDNA Eraser (RR047A, TaKaRa, Kusatsu, Japan). The expression of the 24 AaMADS genes was investigated by qPCR. The primers for these assays (Table S3) were designed using Primer 5.0, and qPCR was performed using a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and SYBR Green PCR Master Mix (RR420A, TaKaRa, Kusatsu, Japan) according to the manufacturer’s instructions. AaActin was used as a reference gene. Three biological and three technical replicates were performed for each sample.

4.5. Gene Cloning, Vector Construction, and Plant Transformation Gene cloning was performed by PCR using specific primers (Table S4). To construct the AaMADS-GFP fusion genes, the open reading frame of 17 AaMADS genes without the stop codon was amplified using PCR and cloned at the XbaI/BamHI and AgeI/KpnI sites of the pBI121-GFP vector using specific primers (Table S5). The above constructs were confirmed by sequencing and transformed into Agrobacterium tumefaciens strain EHA105 for plant transformation. Transient expression in tobacco (Nicotiana benthamiana) leaves was performed as previously described [25]. Arabidopsis (Columbia ecotype) plants were transformed using the floral dip method [26]. Transgenic Arabidopsis plants were Int. J. Mol. Sci. 2021, 22, 9362 11 of 12

selected on 1/2 strength Murashige and Skoog (MS) medium containing 30 µg mL−1 kanamycin. Expression of AaMADS genes in the transgenic Arabidopsis was assessed by semi-quantitative RT-PCR analyses. The T3 generation was used for the phenotypic analyses. Arabidopsis seeds were treated at 4 ◦C for 2 days and then grown on 1/2 MS medium under long-day conditions (16 h light/8 h dark) at 22 ◦C for 10 days before being transplanted into soil. The light intensity of the growth chambers was 150 µE m−2s−1.

4.6. Subcellular Localization The tobacco leaf epidermis was visualized using confocal laser scanning microscopy (CLSM; Nikon, A1, Tokyo, Japan). The nucleus was labeled using a DAPI (40,6-diamidino- 2-phenylindole) dye. GFP and DAPI signals were detected under 500–530 and 420–480 nm emission ﬁlters, respectively.

5. Conclusions A. amurensis is a perennial plant that flowers under natural conditions at extremely low temperatures, which makes it a potential model for investigating the effects of temperature on flowering regulation in other angiosperms. Among the TFs involved in flowering regulation, the MADS-box genes stand out for their central role. Based on transcriptomic and phylogenetic analyses, we found 43 novel MIKCc-type MADS-box genes involved in the regulation of organogenesis and floral development in A. amurensis, including genes involved in the ABCDE model, SVP and SOC1. Moreover, heterologous expression in Arabidopsis of three of these SOC1-like genes was shown to induce early flowering, suggesting their critical role in the regulation of flowering time in A. amurensis. Further, loss-of-function, overexpression, and ectopic expression studies are needed to elucidate the processes regulated by each of these MADS-box genes, as well as the particular features of flowering regulation in A. amurensis.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/ijms22179362/s1. Author Contributions: Conceptualization, S.G. and A.Z.; investigation, L.R., H.S., S.D. and A.Z.; resources, J.W. and A.Z.; data curation, S.F., K.Q. and J.W.; writing—original draft preparation, L.R., H.S. and A.Z.; writing—review and editing, S.G. and A.Z.; funding acquisition, A.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Postdoctoral Science Foundation of Heilongjiang Province of China (LBH-Q19004), the National Natural Science Foundation of China (31902052), Natural Science Foundation of Heilongjiang Province of China (YQ2020C006). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All the raw sequencing reads for all the samples have been submitted to the National Centre for Biotechnology Information (NCBI) Gene Expression Omnibus with accession number GSE126456. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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