HORTSCIENCE 51(6):664–668. 2016. PrseAG displayed extremely early flowering, bigger stamens and carpels, and homeotic conversion of petals into staminoid organs, Molecular Characterization and but ectopic expression of PrseAG-1 could not mimic the phenotypic ectopic expression of Functional Analysis of an PrseAG in Arabidopsis (Liu et al., 2013). The legume Medicago truncatula contains three AGAMOUS-like Gene CiAG from Pecan C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA- Jiyu Zhang1, Min Wang1, Zhenghai Mo, Gang Wang, like gene (MtSHP). MtAGa and MtAGb were and Zhongren Guo2 expressed early in the floral meristem, and in Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, the third and fourth floral whorls during floral development. In contrast, MtSHP expression Nanjing 210014, China appears late during floral development Additional index words. Carya illinoinensis, MADS-box, CiAG, overexpression, flowering, (Serwatowska et al., 2014). On the basis of transgenic Arabidopsis the highly conserved domain of MADS, ho- mologues of AG have allowed cloning from Abstract. The floral homeotic C-function gene AGAMOUS (AG) has been shown to be a number of species (Zahn et al., 2006) such critical in the determination of stamen and carpel identity in Arabidopsis. In the present as apple (Van der Linden et al., 2002), study, a new homologue of AGAMOUS gene from pecan [Carya illinoinensis (Wangenh.) grapevine (Boss et al., 2001), black cherry K. Koch], denoted by CiAG, was isolated and its function was characterized. The (Liu et al., 2010), and japanese apricot (Hou complementary DNA (cDNA) of CiAG contains an open reading frame of 687 base pairs et al., 2011). (bp) encoding 227 amino acids. Multiple sequence comparisons revealed that CiAG had Pecan (C. illinoinensis), which belongs to the typical MIKC structure. Phylogenetic analysis indicated that CiAG is closely related the Juglandaceae family, is of great nutri- to C-lineage AG. The expression of CiAG was highly accumulated in the reproductive tional value (Hal, 2000). It originates from tissues (staminate flowers, pistillate flowers, and fruitlets) than in vegetative tissues the northern United States and has been (leaves and current-growth branches). Arabidopsis overexpressing CiAG exhibited introduced in China for 100 years. The earlier flowering. The homeotic transformations of petals into stamen organs were flower of pecan is unisexual; staminate observed in 35S::CiAG transgenic plants. All these results indicated that CiAG plays flower consists of a calyx and androecium, a key role in the process of flower development of pecan. whereas the pistillate flower has a calyx and a pistil; both of them have no petals. The cultivars of pecan can be divided into two A number of MADS-box genes are in- fungi, plants, and animals (Alvarez-Buylla categories: protogynous, such as ‘Mahan’, volved in the control of the development and et al., 2000; Bodt et al., 2003; Garc´ıa-Maroto ‘Kanza’, and ‘Posey’; and protandrous such specification of flower organs in higher et al., 2003). All the ABCDE MADS-box as ‘Pawnee’, ‘Osage’, and ‘Canton’ (Reid plants. In the well-known ABC model (Coen genes are of the MIKC type with regard to the and Hunt, 2000; Wood et al., 1997). As and Meyerowitz, 1991), three classes of presence of four distinct domains, which are C-function MADS-box gene plays a key role genes, A, B, and C functions, specify the a highly conserved MADS-box (M) domain, both in stamen and carpel formation, we four organ types of the typical angiosperm an intervening (I) domain, a moderately con- isolated a cDNA sequence of CiAG in this flower. According to this model, sepal iden- served keratin (K) domain, and a C-terminal study. The expression profiles of CiAG gene tity is specified by the A function alone, petal (C) domain (Theissen et al., 2000). in reproductive tissues (staminate flowers, formation is controlled by A and B functions, AG, a class C gene, is required for normal pistillate flowers, and fruitlets) and vegeta- stamen development is regulated by the development of third and fourth whorl floral tive tissues (leaves and current-growth combination of B and C functions, and carpel organs. In the third whorl, AG functions in the branches) of three varieties (‘Shaoxing’, formation is determined by C function alone. specification of stamens; in the fourth whorl, ‘Pawnee’, and ‘Mahan’), were investigated It should be noted that there exists an antag- AG is required for specification of carpels and by quantitative real-time reverse transcrip- onistic interaction between A and C func- provision of determinacy to the floral meri- tion polymerase chain reaction (qRT-PCR). tions. With the isolation of D function for stem (Martin et al., 2006; Sieburth et al., We then transformed the CiAG gene into ovule development (Angenent et al., 1995) 1995). Arabidopsis to study the function of CiAG on and E function (Theissen, 2001), which is Ectopic expression of GmGAL2 (Gly- flowering. required for the specification of petals, sta- cine max AGAMOUS Like 2)inArabidopsis mens, and carpels, the ABC model has been enhances flowering, under both long-day Materials and Methods extended to the ABCDE model. All the genes and short-day conditions, by promoting belonging to the ABCDE model are members the expression of key flowering genes, Plant materials. The grafting seedlings of of the MADS-box gene family except for CONSTANS (CO) and FLOWERING LOCUS pecan were grown in Nanjing, Jiangsu prov- APETALA2 (AP2) genes (Theissen et al., T (FT), and lowering the expression of floral ince, China. Staminate flowers, pistillate 2000). MADS-box genes encoding homeotic inhibiter FLOWERING LOCUS C (FLC) (Xu flowers, fruitlets, leaves, and current-growth transcription factors are a highly conserved et al., 2010). HpAG,aHosta plantaginea branches were collected from the 6-year-old gene family and these genes widely exist in Aschers AGAMOUS homologous gene, iso- cultivars of Mahan, Shaoxing, and Pawnee in lated from developing flowers, plays a crucial May. The tissues were collected from the role in stamen specification and gynoecium same direction of each tree, three trees as development (Wang et al., 2012). Overex- three replicates. Tissues were immediately Received for publication 2 Dec. 2015. Accepted for pression of a Brassica rapa MADS-box frozen in liquid nitrogen and stored at –80 °C publication 16 Mar. 2016. gene, BrAGL20, induces early flowering until used. This work was supported by the National Natural time phenotypes in Brassica napus (Hong Cloning of CiAG. Total RNA was ex- Science Foundation of China (Grant nos. 31200502 et al., 2013). Two transcript isoforms of tracted from the staminate flowers of ‘Mahan’ and 31401854), and the Natural Science Founda- AGAMOUS homologues, PrseAG (Prunus tion of Jiangsu Province (Grant no. BK20150552). using The Plant Total RNA Extraction Kit 1Jiyu Zhang and Min Wang contributed equally to serrulata AGAMOUS) from single and (BioTeke, Beijing, China). First-strand this work. PrseAG-1 from double flower Prunus lannesi- cDNA was synthesized from 1 mgtotal 2 Corresponding author. E-mail: zhongrenguo@ ana, respectively, showed different functions. RNA with an oligo(dT)18 adaptor primer cnbg.net. The transgenic Arabidopsis containing 35S:: using PrimeScript RTase (TaKaRa, Japan). 664 HORTSCIENCE VOL. 51(6) JUNE 2016 | BREEDING,CULTIVARS,ROOTSTOCKS, AND GERMPLASM RESOURCES We have obtained eleven highly conserved and VvAG-R (5#-CGCCATAACAGGGCAA AGL11 (NP_192734), SEP1 (NP_568322), MADS domain amino acid motifs from pecan TAACCT-3#), and the PCR product was cloned SEP2 (NP_186880), SEP3 (NP_850953), previously. In this study, we cloned the full- and sequenced. and SEP4 (NP_849930). length cDNA of the fragment 1 which belonged Amino acid alignment and phylogenetic Gene expression analysis of CiAG in to AGAMOUS group (Mo et al., 2013). The analysis. The deduced amino acid sequences pecan using qRT-PCR. Total RNA isolation gene-specific primers GSP1 (5#-CACTAC were analyzed in the NCBI BLAST pro- was done as described above. Reverse tran- TAATCGTCAAGTCACCTTCTGT-3#) and gram (http://www.ncbi.nlm.nih.gov) for scription was performed using 1 mg of total GSP2 (5#-CTTCTGTAAGAGGCGCAA searching the protein sequences of homo- RNA with the ReverTra Ace qPCR RT Kit CGGCTT-3#) were designed based on the logues. Alignment of deduced amino acid (TOYOBO, Code No. FSQ-101) according to conserved MADS domain nucleotide se- sequences was performed by using the the manufacturer’s instructions. The gene- quence. Nested PCR was carried out to Clustal W multiple alignment program of specific primers for CiAG were QTAG-F: 5#- isolate the 3# end of the CiAG gene. The the BioEdit software. A phylogenetic tree AGGCTGCTACTCTACGACAAC-3# and gene-specific primer GSP1 and an abridged was constructed using the neighbor-joining QTAG-R: 5#-GGTTCCTCTCATTCTCCGC universal amplification primer AUAP (5#- method in the MEGA 5.1 software with TATC-3#.TheCiActin gene was used as a GGCCACGCGTCGACTAGTAC-3#)were 1000 replication bootstrap tests. The acces- positive internal control with primers ACTIN-F used for first-round PCR, and the gene- sion numbers of the protein sequences used (5#-GCTGAACGGGAAATTGTC-3#), and specific primer GSP2 (5#-CTTCTGTAA in this study are as follows: Juglans regia, ACTIN-R (5#-AGAGATGGCTGGAAGAGG-3#). GAGGCGCAACGGCTT-3#) and AUAP JrAG (CAC38764); Corylus heterophylla, qRT-PCR was carried out on the Applied were used for second-round PCR using the ChMADS1 (AEU08497); Prunus persica, Biosystems 7300 Real Time PCR System with first-round PCR product. The cycling pro- PpMADS4 (AAU29513); Momordica char- a20-mL reaction volume, containing 1 mL gram consisted of an initial denaturation antia, McMADS2 (ABC25564); Citrus 10-fold diluted cDNA, 0.3 mL(10pM)ofeach at 94 °C for 5 min, followed by 35 cycles sinensis, CsAG (ADP02394); Prunus sero- primer, 10 mL SYBRÒ Premix Ex Taqä of 94 °C for 30 s, 65 °C for 30 s, 72 °Cfor tina, PsAG (ACH72974); Jatropha curcas, (Perfect Real Time) (TaKaRa Code: DRR041A), 1 min, and a final extension at 72 °Cfor JcAG (AEA11211); Gossypium hirsutum, and 8.4 mL sterile double-distilled water.