agronomy Article Isolation and Characterization of the PISTILLATA Ortholog Gene from Cymbidium faberi Rolfe Yue Fei and Zhi-Xiong Liu * College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China * Correspondence: [email protected]; Tel.: +86-716-8066260 Received: 29 May 2019; Accepted: 1 August 2019; Published: 2 August 2019 Abstract: Cymbidium faberi Rolfe is a very popular potted plant in China, Japan and Korea where it has been cultivated for centuries. The economic value of this popular native Asian orchid could be enhanced by changes in its floral traits. In Arabidopsis, PISTILLATA (PI) is involved in regulating petal and stamen development. In order to investigate the possible role of the PI ortholog involved in floral development, we isolated CyfaPI from C. faberi. Protein alignment and a phylogenetic tree grouped CyfaPI in the PI lineage. CyfaPI transcripts were detected in all floral organs, but were absent in leaves. Moreover, in flowers, the highest expression level of CyfaPI was present in the gynostemium and the lowest level was found in anther caps. In addition, ectopic expression of CyfaPI in Arabidopsis pi-1 mutant rescued petal development, and complement the development of filament-like structure (part of stamen), but failed to complement anther development in the stamen whorl. All these finding suggest that CyfaPI is mainly responsible for perianth and gynostemium development in C. faberi. Our data may help to trace the development of the gynostemium program and evolution in orchids. Keywords: PISTILLATA; Cymbidium faberi; MADS-box gene; Floral development; Orchidaceae 1. Introduction Cymbidium faberi Rolfe is a very popular potted plant in several countries of East Asia, and has been cultivated for centuries in China, Japan and Korea. Many artificially propagated varieties have been developed based on various flower traits including their color and number as well as the form of the perianth (sepal and petal). All of these elements contribute to the visual appreciation of orchids. The perianth is the most visually obvious structure, which is related to its primarily function as an optical attraction for pollinators. Moreover, the molecular mechanisms underlying the perianth identity program have been intensively studied in model species such as Arabidopsis thaliana, Antirrhinum majus and Petunia hybrida [1], and the evolution of the perianth identity has been extensively discussed recently in [2]. However, the perianth in monocots does not completely correspond to the obvious differentiation of sepal and petal whorls in model eudicots. In eudicots, the conserved function of the PISTILLATA (PI)/GLOBOSA (GLO) orthologs, such as MtPI from Medicago truncatula [3], TrPI from Taihangia rupestris [4], CabuPI from Catalpa bungei [5], is the regulation of petal and stamen development. Furthermore, each ortholog is able to fully complement the floral phenotype of the Arabidopsis pi mutant, and the expression pattern of each corresponds to their conserved function. In basal angiosperm, PI-like genes usually show broad expression zones in floral organs. For example, HoPI_1 and HoPI_3 from Hedyosmum orientale are expressed in all floral organs including the bracts and carpels. In addition, HoPI_1 mimics the endogenous Arabidopsis PI and fully rescues both petal and stamen development in Arabidopsis pi-1 mutants, while HoPI_3 only rescues petal development, and partially rescues stamen development [6]. In another basal angiosperm Magnolia wufengensis, the PI ortholog MawuPI was only able to rescue filament-like structures in the third whorl, but failed to rescue petal development in Agronomy 2019, 9, 425; doi:10.3390/agronomy9080425 www.mdpi.com/journal/agronomy Agronomy 2019, 9, 425 2 of 11 Agronomy 2019, 20 FOR PEER REVIEW 2 the second whorl [7]. Together, these data provide interesting evidence of a B-function evolutionary Together, these data provide interesting evidence of a B-function evolutionary scenario among scenario among early-diverging clades of the angiosperm. early-diverging clades of the angiosperm. MonocotsMonocots are are one one of theof the major major clades clades of angiosperm,of angiosperm, and and the the Orchidaceae Orchidaceae family family is oneis one of of the the most species-richmost species-rich in this clade;in this it clade; includes it includes species species with highly with highly diversified diversified and specialized and specialized floral floral shapes. Previousshapes. studies Previous have studies indicated have thatindicated floral that diversity floral diversity in orchids in results orchids from results changes from changes in the regulatory in the regionsregulatory of the regionsMADS -boxof the genes MADS followed-box genes by followed gene duplication by gene duplication events [8], events and that [8],PI and-like that genes PI-like work togethergenes withwork ditogetherfferent AP3with/AGL6 different homologs AP3/AGL6 involved homologs in specifying involved sepalin specifying/petal/lip sepal/petal/lip development in Oncidiumdevelopmentand Phalaenopsis in Oncidiumorchids and Phalaenopsis [9]. Here, we orchids isolated [9]. aHere,PI ortholog, we isolatedCyfaPI, a PIfrom ortholog, a Chinese CyfaPI, traditional from orchida ChineseC. faberi traditionalRolfe. The orchid flower C. offaberiC. faberiRolfe.consists The flower of three of C. whorls faberi consists of floral of organs, three whorls with three of floral sepals in whorlorgans, 1, with two three petals sepals and ain lip whorl in whorl 1, two 2, petals and a gynostemiumand a lip in whorl fused 2, and by thea gynostemium male and female fused tissues by in whorlthe male 3 (Figure and female1). CyfaPI tissuestranscripts in whorl 3 (Figure are detected 1). CyfaPI in all transcripts floral organs, are detected but are in absent all floral in theorgans, leaves, whichbut showedare absent a di infferent the leaves, expression which pattern showed to PIa -likedifferent genes expression from eudicots. pattern Further to PI-like functional genes analysisfrom revealedeudicots. that FurtherCyfaPI functionalis able to analysis fully complement revealed that petals CyfaPI in is whorl able to 2, fully but cancomplement only rescues petals filament-like in whorl structures2, but can in whorlonly rescues 3 of flowers filament-like in transformed structuresArabidopsis in whorl 3 pi-1 of flowersmutants. in Ourtransformed data may Arabidopsis provide the pi-1 clue to helpmutants. trace Our the data gynostemium may provide development the clue to help program trace andthe gynostemium evolution in orchids.development program and evolution in orchids. Figure 1. Flower and inflorescence of Cymbidium faberi. (A) Flower of C. faberi;(B) The gynostemium Figure 1. Flower and inflorescence of Cymbidium faberi. (A) Flower of C. faberi; (B) The gynostemium and ovary of C. faberi;(C) The inflorescence of C. faberi. Sepals (sep), petals (pet), lips (lip), anther cap and ovary of C. faberi; (C) The inflorescence of C. faberi. Sepals (sep), petals (pet), lips (lip), anther cap (anc),(anc), gynostemium gynostemium (gyn), (gyn), ovary ovary (ova). (ova). Bars:Bars: 5 mm. 2. Results 2. Results 2.1. Cloning and Sequence Alignments of CyfaPI 2.1. Cloning and Sequence Alignments of CyfaPI The CyfaPI (Cymbidium faberi PISTILLATA) cDNA is 932 bp with a 633 bp ORF (Open Reading The CyfaPI (Cymbidium faberi PISTILLATA) cDNA is 932 bp with a 633 bp ORF (Open Reading Frame) encoding a 210 amino acid (aa) protein (Genbank accession number: MK450600.1). Putative Frame) encoding a 210 amino acid (aa) protein (Genbank accession number: MK450600.1). Putative proteinprotein alignment alignment and and a a phylogenetic phylogenetic analysis analysis grouped grouped CyfaPI CyfaPI into into the theGLO/PI GLO lineage/PI lineage (Figure (Figure 2). In 2). In addition,addition, CyfaPI CyfaPI is is nested nested within within a aclade clade of of orthologs orthologs from from other other monocots monocots and and is most is most closely closely relatedrelated to orthologsto orthologs from from the the Orchidaceae, Orchidaceae, suchsuch as Oncidium OMADS8OMADS8 (97.14% (97.14% identity) identity) (Figure (Figure 2A).2A). ComputationalComputational translation translation showsshows thatthat CyfaPI is is a aputative putative transcriptional transcriptional regulator regulator that thatcontains contains a a highlyhighly conserved 57 57 aa aa MADS-box MADS-box domain domain (1–57) (1–57) at the atN-terminal the N-terminal region, a region, less conserved a less conserved64 aa K 64domain aa K domain (87–150) (87–150) in the middle in the region middle and region a highly and conserved a highly 16 aa conserved PI motif (193–208) 16 aa PI in motif the variable (193–208) in theC terminal variable region C terminal (151–210). region Moreover, (151–210). CyfaPI Moreover, contains CyfaPI three contains putative three amphipathic putative amphipathicα-helices, α-helices,K1(87–108), K1(87–108), K2 (121–135) K2 (121–135) and K3 (143v167) and K3 (143v167) subdomains subdomains that could that potentially could potentially mediate protein mediate proteininteraction interaction (Figure (Figure 2B) [10–12].2B) [ 10 In– 12addition,]. In addition, CyfaPI is CyfaPI 55.56%, is 55.66%, 55.56%, 59.05%, 55.66%, 60.85%, 59.05%, 71.09%, 60.85%, 64.29%, 71.09%, 64.29%,74.53%, 74.53%, 79.05%, 79.05%, and 67.14% and 67.14% identified identified to PI tofrom PI from A. thalianaA. thaliana, TrPI, TrPIfrom from T. rupestrisT. rupestris, MtPI, MtPI from from M. M. truncatulatruncatula, CabuPI, CabuPI