Planta (2015) 241:387–402 DOI 10.1007/s00425-014-2190-3

ORIGINAL ARTICLE

Distinct subfunctionalization and neofunctionalization of the B-class MADS-box genes in Physalis floridana Shaohua Zhang • Ji-Si Zhang • Jing Zhao Chaoying He

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Received: 8 August 2014 / Accepted: 2 October 2014 / Published online: 19 October 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Main conclusion This work suggested that in Physalis PFGLO1–PFDEF primarily determined corolla and androecium identity, and acquired a novel role in gynoecia functionality, while PFGLO2–PFTM6 functioned in pollen maturation only. Abstract The B-class MADS-box genes play a crucial role in determining the organ identity of the corolla and androecium. Two GLOBOSA-like (GLO-like) PFGLO1 and PFGLO2 and two DEFICIENS-like (DEF-like) PFDEF and PFTM6 genes were present in Physalis floridana. However, the double-layered-lantern1 (doll1) mutant is the result of a single recessive mutation in PFGLO1, hinting a distinct divergent pattern of B-class genes. In this work, we utilized the tobacco rattle virus (TRV)-mediated gene silencing approach to further verify this assumption in P. floridana. Silencing of PFGLO1 or/and PFDEF demonstrated their primary role in determining corolla and androecium S. Zhang and J.-S. Zhang contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2190-3) contains supplementary material, which is available to authorized users. S. Zhang  J.-S. Zhang  J. Zhao  C. He (&) State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Beijing 100093, China e-mail: [email protected] S. Zhang  J.-S. Zhang  J. Zhao University of Chinese Academy of Sciences, Yuquan Road 19, Beijing 100049, China J.-S. Zhang Anshan Normal University, Pingan Street 43, Anshan 114000, China

identity. However, specific PFGLO2 or/and PFTM6 silencing did not affect any organ identity but showed a reduction in mature pollen. These results suggested that both PFGLO2 and PFTM6 had lost their role in organ identity determination but functioned in pollen maturation. Evaluation of fruit setting in reciprocal crosses suggested that both PFGLO1 and PFDEF might have acquired an essential and novel role in the functionality of gynoecia. Such a divergence of the duplicated GLO–DEF heterodimer genes in floral development is different from the existing observations within Solanaceae. Therefore, our research sheds new light on the functional evolution of the duplicated B-class MADS-box genes in angiosperms. Keywords MADS-box gene  Functional evolution  Fertility  Organ identity  Physalis Abbreviations BiFC Bimolecular fluorescence complementation cDNA Complementary DNA GFP Green fluorescence protein ORF Open reading frame TRV Tobacco rattle virus VIGS Virus-induced gene silencing YFP Yellow fluorescence protein

Introduction MADS-box genes encode a large family of putative transcription factors, which function in the evolution and development of flowers (Theißen and Saedler 2001). Most components of the well-established floral ABC and floral

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quartet models that depict the determination of floral organ identity are MADS-domain proteins (Coen and Meyerowitz 1991; Theißen and Saedler 2001). Among these, the B-class MADS-box genes are essential for organ identity of the corolla and androecium. Two closely related paralogs GLOBOSA (GLO)/PISTILLATA (PI) and DEFICIENS (DEF)/APETALA3 (AP3) genes are present in Antirrhinum and Arabidopsis, respectively (Sommer et al. 1990; Jack et al. 1992; Tro¨bner et al. 1992; Goto and Meyerowitz 1994; Lamb and Irish 2003). They form obligate heterodimers in Antirrhinum and Arabidopsis (Winter et al. 2002), and any functional mutation of each paralog produces the obvious floral homeotic mutants wherein the corolla is transformed to calyx, and the androecium is replaced by gynoecium (Sommer et al. 1990; Jack et al. 1992; Tro¨bner et al. 1992; Goto and Meyerowitz 1994). However, one cannot replace another to accomplish the reciprocal complementation of the mutants. The developmental system is robust and evolvable in flowering plants (Lamb and Irish 2003; Geuten et al. 2011). Both GLO and DEF gene lineages were duplicated before the ancestor of the core asterids (Hernandez–Hernandez et al. 2007; Viaene et al. 2009); thus, resulting in GLO1- and GLO2-clades in the GLO lineage, and DEFand TOMATO MADS6 (TM6)-clades in the DEF lineage. Duplication of genes often provides a basis for diversification in either molecules or morphology. The diversification usually accompanies the definite fate of duplicated genes that either acquire a new role or undergo subfunctionalization. The two duplicates for each lineage of B-class MADS-box genes were found to redundantly partition the role of the B-function with a variable subfunctionalization process in Petunia, Nicotiana and Solanum (Vandenbussche et al. 2004; de Martino et al. 2006; Rijpkema et al. 2006; Geuten and Irish 2010). Thus, they contribute to the morphological diversity and adaptation evolution of the perianth within the Solanaceae. Physalis is a genus of the family of Solanaceae. Unlike Petunia, Nicotiana and Solanum, the persistent floral calyx outgrows into a balloon-like fruiting calyx, which encapsulates the berry. This novel morphology of the fruiting calyx is named as the ‘‘Chinese lantern’’ or inflated calyx syndrome (He et al. 2004), which is of key research interest to us. Our work highlights Physalis as a new ‘model plant’ for the study of evolution, development, and ecology (He and Saedler 2005, 2007; He et al. 2007; Wang et al. 2012; Zhang et al. 2014a, b). Analyses of the double-layered lantern1 (doll1) mutant revealed that two GLO genes including PFGLO1 and PFGLO2 are present in P. floridana (Zhang et al. 2014a). However, PFGLO1 and PFGLO2 have diverged in terms of their molecular interactions and developmental roles, implicating a new divergent pattern of the GLO duplicates within the Solanaceae.

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Nonetheless, the function of the PFDEF and PFTM6 in the DEF lineage is not yet characterized in Physalis. In the present study, we attempted to gain a comprehensive understanding of the developmental role and the functional diversification of B-class MADS-box genes in Physalis. Thus, we used the tobacco rattle virus (TRV)-mediated gene silencing approach to infer the developmental role of PFDEF and PFTM6. We found that PFGLO1 or/and PFDEF are essential for organ identity of corolla and androecium, and have acquired a new role in functionality of gynoecia. However, PFGLO2 or/and PFTM6 have a special role in pollen maturation. Our work revealed a new function divergent pattern of the duplicated B-class genes within Solanaceae; thus, providing a new insight into the functional evolution of the duplicated genes in general.

Materials and methods Plant materials Physalis floridana P106 (He and Saedler 2005) was grown in a growth chamber under long day conditions (16/8 h light/dark cycle) with a constant temperature of 22 °C. Floral organs were harvested for total RNA isolation. mRNA in situ hybridization A complementary DNA (cDNA) fragment covering a portion of the C-domain and part of the 30 untranslated region (30 UTR) from each of PFDEF (283 bp), or PFTM6 (406 bp) was used as a probe template. Probes were synthesized using the T7 RNA polymerase driven by a T7 promoter and labeled with digoxigenin (DIG) using the DIG-RNA-labeling-kit (Roche, Mannheim, Germany). The paraffin sections were prepared under RNase-free conditions and were deparaffinized and rehydrated. Hybridizations were performed at 45 °C overnight and the slides were washed at a temperature of 50 °C. Hybridization signal was detected by chemiluminescence with NBT/ BCIP stock solution (Roche). Images were examined under a fluorescence microscope (Axioskop40 with HBO100, Zeiss). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from young floral buds using Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized with SuperScript III Reverse Transcriptase (Invitrogen) using the Poly (T) 17 primer. The qRT-PCR was performed using the SYBR Premix Ex Taq (Perfect Real Time) kit (TaKaRa, Dalian,

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China) according to the manufacturer’s manual of the Mx3000p Real Time system (Stratagene). The amplification conditions were: 30 s at 95 °C, 1 cycle, followed by 40 cycles of 5 s at 95 °C, 20 s at 60 °C. Data was monitored to detect dissociation curves. PFACTIN was used as the housekeeping gene, and relative quantification was performed using the previous described method (Livak and Schmittgen 2001).

Zeiss microscope (Zeiss). For scanning electronic microscopy (SEM) analyses, fresh materials were fixed in formalin–acetic–alcohol solution (95 % ethyl alcohol: glacial acetic acid: formaldehyde, 8:1:1, by vol.), sputter-coated with gold, and examined with a digital scanning microscope (Hitachi S-4800).

Transient protein expression assays

Emasculation was done before self-pollination either in wild-type or VIGS flowers. To evaluate the pollen, pollen from flowers from each VIGS line was used to pollinate wild-type stigma. Conversely, stigma from each VIGS line was pollinated using wild-type pollen to evaluate the effects of gene downregulation on female flowers. Crosses in wild type were used as control. In each case, 40 pollinations were performed. The fruit-set number, berry weight, and seed number per berry were determined statistically. The correlation coefficient (r) was evaluated using the CORREL program. Significance evaluation (P value) was evaluated using the two-tailed Student’s t test. The mean and standard deviation are presented.

For subcellular localization studies, the open reading frame (ORF) of PFDEF or PFTM6 was cloned into the Super1300 expression vector using the Xba I/Kpn I (TaKaRa) restriction sites and fused to the green fluorescence protein (GFP). For bimolecular fluorescence complementation (BiFC) assays, the ORFs of PFGLO1, PFGLO2, PFDEF, PFTM6, MPF2, MPF3, PFAG, PFSEP1 and PFSEP3 were, respectively, cloned into the pSPYNE-35S and pSPYCE-35S vector pair using the Xba I/BamH I (TaKaRa) restriction sites. These paired vectors were designed to express either the N- or C-terminal halves of the yellow fluorescence protein (YFP). The recombinant GFP constructs or the construct combination of two proteins fused with the N- or C-terminal halves of YFP were agroinfiltrated into the leaf epidermal cells of Nicotiana benthamiana (Walter et al. 2004). The fluorescence signal of the GFP or YFP was detected 48 h after injection using a confocal laser scanning microscope (Olympus FV1000MPE).

Crosses and trait quantifications

Sequencing analyses The primers for probe preparation, VIGS construct creation and qRT-PCR analyses used in the present work are shown in Table S2. The VIGS constructs made were verified via sequencing analyses.

Virus-induced gene silencing (VIGS) and genotyping

Results

VIGS procedures were performed according to previously described methods and conditions (Zhang et al. 2014b). A 479-bp cDNA fragment of PFGLO1, a 416-bp cDNA fragment of PFGLO2, a 422-bp cDNA fragment of PFDEF, a 406-bp cDNA fragment of PFTM6, and the tandemly repeated fragment of each of the two combinations were transformed into the TRV2 binary vector for the creation of VIGS constructs for both single and double gene silencing. The used fragment is gene specific (Fig. S1), thus guaranteeing the specificity of gene silencing. The number of the VIGS plants is shown in Table S1. The gene silencing in VIGS-infected flowers was confirmed using qRT-PCR analysis.

Fine expression of PFDEF and PFTM6 genes

Morphological analyses Pollen maturation was detected using the iodine–potassium iodide (I2–KI) staining. The floral buds, mature flowers, androecia, and pollen grains were photographed using a

We first investigated the expression of PFDEF and PFTM6 by in situ mRNA hybridization. Antisense PFDEF-specific probing showed that PFDEF was expressed in the floral meristem and primordium of the corolla and androecium; however, the expression weakened upon organ formation (Fig. 1a–c). Antisense PFTM6-specific probing demonstrated that PFTM6 was expressed in floral meristem and the primordium of the corolla and androecium (Fig. 1d–f). Apparently, PFTM6 shared an identical expression domain with PFDEF during floral organ formation. However, PFTM6 was mainly expressed in androecium and gynoecium in the later developmental stages (Fig. 1g). Hybridizations with sense probes of PFTM6 (Fig. 1h) or PFDEF (Fig. 1i) were used as the controls. qRT-PCR showed that PFTM6 was also highly expressed in the fruiting calyces and the developing berries (Fig. S2). These results suggest different roles for these two genes in the flower/fruit

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Fig. 1 mRNA in situ hybridizations of the DEF lineage genes in floral organ initiation of Physalis floridana. a–c Antisense probe of PFDEF. d–g Antisense probe of PFTM6. h Sense probe of PFTM6. i Sense probe of PFDEF. cal calyx primordial, cor corolla primordial, and androecium primordial, gyn gynoecium primordial, fm floral meristem. In g, they symbolize the corresponding organs. Bars 100 lm

development of Physalis. Next, we exploited the VIGS method to reveal the function of PFDEF and PFTM6. Mutational phenotype as a result of single PFDEF downregulation Altogether, 116 P. floridana plants were infiltrated with the Agrobacterium containing the PFDEF-VIGS constructs, and 65 of the resulting PFDEF-VIGS plants showed similar and visible floral variation (Table S1). In comparison to the wild type (Fig. 2a, b), some flowers from these VIGS plants were apparently normal as wild type, termed Wt0. Nevertheless, three categories of mutated phenotypes related to homeotic transformation of both corolla and androecium into the calyx and gynoecium (m1, m2, and m3) were observed in these lines (Fig. 2c–k). In grade m1, the corolla was completely mutated to the calyx but the

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androecium was normal (Fig. 2c, d); while in grade m2, the androecium was partially transformed to gynoecium (Fig. 2e–g). The corolla and androecium were completely transformed to calyx and gynoecium, respectively, in the most severe category m3 (Fig. 2h–k). The transformed gynoecium was either fused with or separated from the original gynoecium (Fig. 2i, k). The transformation of the corolla to the calyx was further verified through SEM analyses of the epidermal cells. The cells on the abaxial or adaxial surface of the mature wild-type calyx were lobate and imbedded with trichomes (Fig. 2l, m). The cells on abaxial of the mature wild-type corolla were undifferentiated regular cells with trichomes developed while the cells on adaxial surface of corolla were regular dome cells (Fig. 2n, o). However, the cells on abaxial or adaxial surface of the mutated second floral whorl in the PFDEFVIGS flowers were similar to those of the wild-type calyx (Fig. 2p, q); thus, further supporting the homeotic transformation of the corolla to the calyx in the PFDEF-VIGS flowers. Total RNA of four flowers from 8 PFEDF-VIGS plants was subjected to qRT-PCR analyses to correlate the phenotypic variation with the extent of PFDEF downregulation. PFDEF expression was knocked down to differing extents in the m1, m2, and m3 flowers as compared to WT, while it was not significantly altered in Wt0 (Fig. 2r). The extent of the phenotypic transformation apparently depended on the dose of PFDEF mRNA. Grade m3 showed the most severe phenotypic variations, wherein PFDEF expression was around 10 % of that observed in the wild type (Fig. 2r). PFTM6 controls pollen maturation A total of 75 out of 112 PFTM6-VIGS lines were produced (Table S1). The results of qRT-PCR analyses showed that the expression of PFTM6 was specifically and efficiently knocked down, as evidenced by the fact that PFTM6 expression in some flowers was less than 10 % of that seen in the wild type (Fig. S3). Even the most severe downregulation of PFTM6 did not reveal any visible phenotypic variation in floral organs (Fig. 2s, t). We performed I2–KI staining assays to reveal any potential defect in pollen development. Around 94 % of wild-type pollen grains were deeply stained blue (Fig. 2u), while less than 50 % of the pollen grains from the PFTM6-VIGS flowers were stained blue (Fig. 2v). Since, mature pollen stains blue, less than 50 % pollen grains stained indicated that the maturation was impaired as a result of severe PFTM6 downregulation. In one extreme case, only 10.5 % of the pollen grains assayed were mature (Fig. 2w). The extent of PFTM6 downregulation seemed to negatively correlate to pollen maturation (Fig. 2w, x). Therefore, downregulation of PFTM6

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Fig. 2 Tobacco rattle virus (TRV)-mediated silencing of the DEF lineage genes in P. floridana. a, b The wild-type (WT) flower. The PFDEF-VIGS flowers. c, d Grade m1 of the PFDEF-VIGS flowers. Homeotic variation only occurred in corolla that was completely transformed into calyx. e–g Grade m2 of the PFDEF-VIGS flowers. Homeotic variation occurred in both corolla and androecia. Part of androecia was transformed into gynoecia. h–k Grade m3 of the PFDEF-VIGS flowers. Corolla and androecia were transformed into calyx and gynoecium, respectively. SEM of the first and the second floral whorl. l Abaxial epidermal cells of WT calyx. m Adaxial epidermal cells of WT calyx. n Abaxial epidermal cells of WT corolla. o Adaxial epidermal cells of WT corolla. p Abaxial epidermal cells of the second whorl of the grade m3 in the PFDEF-VIGS flowers. q Adaxial epidermal cells of the second floral whorl of the grade m3 in the PFDEF-VIGS flowers. r The relative PFDEF expression in the flowers from the PFDEF-VIGS plants with different

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grades of wild-type-like (Wt0) and mutated categories (m1, m2, and m3) in comparison with that of wild-type flowers (WT). PFACTIN was used as an internal control. Gene amplification was repeated three times using the total RNA from each categorized flowers of the indicated plants (P15-P91). The mean and standard deviation are shown. PFTM6-VIGS analyses. s, t PFTM6-VIGS flowers. Part of calyx and corolla was removed to show the organs inside in t. u, v I2– KI staining of the wild-type pollen (u) and PFTM6-VIGS pollen (v). The yellow pollen was immature and the blue pollen was mature. w Quantification of mature pollen rate in the WT and four PFTM6VIGS flowers (#1–#4). x The relative PFTM6 expression in the androecia of flowers in w. PFACTIN was used as an internal control. Gene amplification was repeated three times using the total RNA from each flower as indicated. The mean and standard deviation are shown. Bars 2 mm (a–k, s, t), 100 lm (l–q, u, v)

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alone may directly or indirectly block sugar translocation to the pollen, thereby preventing the maturation of pollen grains.

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lineages (PFDEF and PFTM6) were generated. Since the heterodimers of PFGLO1–PFDEF and PFGLO2–PFTM6 are formed (Zhang et al. 2014a), their genes were next downregulated together in P. floridana.

Double downregulation of PFDEF and PFTM6 PFDEF and PFTM6 are paralogs in the DEF lineage that apparently diverged in function, which is corroborated by the observation that their encoded proteins do not interact physically (Zhang et al. 2014a). We attempted to reveal whether they genetically interacted through generating the PFDEF–PFTM6-VIGS plants. In addition to wild-type flowers (Fig. 3a), some wild-type-like (Wt0) flowers, and three categories of mutated phenotypes were seen in the co-VIGS mutated plants (Fig. 3b–i), which was basically similar to those of the PFDEF-VIGS mutants. The expression of both PFDEF and PFTM6 was analyzed in the four categories of flowers including Wt0, m1, m2 and m3 from the six PFDEF–PFTM6-VIGS plants (Fig. 3j, k). The PFDEF expression was downregulated to a different extent in each category (Fig. 3j), which correlated to the extent of homeotic variation. The PFTM6 transcript accumulation was variable. It was upregulated in some mutated flowers where PFDEF was severely downregulated, while it was downregulated in some flowers where PFDEF was moderately downregulated or its expression remained unchanged (Fig. 3k). These results suggest a possible repression of the PFTM6 expression by PFDEF. Additional phenotypic variation was not observed in the PFDEF–PFTM6-VIGS plants as compared to the PFDEFVIGS flowers (Fig. 2). Apparently, the variation of PFTM6 expression did not affect the organ identity. Wt0 flowers showed no homeotic variation of androecium, as a result, of severe PFTM6 downregulation. Moreover, I2–KI staining assays showed that the pollen maturation in these flowers was severely affected (Fig. 3l–o) to an extent similar to that of PFTM6-VIGS flowers (Fig. 2u–x). In mutated grades, m1, m2 and m3, the androecia had been transformed into normal-looking or deformed gynoecia; thus, no pollen could be detected. However, there was increased accumulation of PFTM6 mRNA transcripts (Fig. 3m) corroborating the notion that PFDEF may repress the expression of PFTM6. Nevertheless, no additional phenotypic variation was seen in the so-called coVIGS flowers in comparison to their single-gene downregulations. Double downregulation analyses of genes between the GLO and DEF lineages To further understand the role of these B-class MADS-box genes in Physalis, double downregulated mutants of genes between the GLO (PFGLO1 and PFGLO2) and DEF

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Downregulated mutants of the GLO–DEF heterodimer genes Similar to single downregulated PFDEF, PFGLO1 is required for organ identity as well (Zhang et al. 2014a). We first analyzed these two genes by downregulating their expression at the same time (Table S1). In comparison to the wild type (Fig. 4a), the floral phenotypic variation in these PFGLO1–PFDEF downregulated double mutants showed a floral homeotic variation (Fig. 4b–g), which resembled their single-gene downregulated mutants. The downregulation of PFGLO1 or/and PFDEF correlated to the phenotypic variation (Fig. 4h, i). In the PFGLO2– PFTM6 double downregulated mutants, no homeotic variation was observed in any floral organs (Fig. 4j, k), but the pollen maturation was severely affected as revealed by I2– KI staining assays (Fig. 4l–n). The pollen maturation negatively correlated to the remaining transcripts of PFGLO2 or/and PFTM6 (Fig. 4n, o). Double downregulating non-heterodimer genes between the two lineages Physical interaction of some B-class MADS-domain proteins was not observed in Physalis; however, their genetic interaction was further analyzed using co-VIGS approach. Forty-two of 84 PFGLO1–PFTM6-VIGS double downregulated plants developed severe homeotic variations (Fig. S4a–d; Table S1) that resembled those of the PFGLO1VIGS flowers (Zhang et al. 2014a). In comparison to WT flowers (Fig. S4a), corolla and androecium were partially or completely transformed into calyx and gynoecium, respectively (Fig. S4b–d). Pollen maturation was severely impaired in the flowers that did not show homeotic variation of androecium into gynoecium (Fig. S4e–g). qRT-PCR confirmed that the PFGLO1 expression showed a 60 % increase over that of the WT, while the PFTM6 expression remained around 10.3–21.4 % of the WT (Fig. S4h). Homeotic variations were different in 60 of 84 PFGLO2–PFDEF-VIGS plants (Table S1). Flowers featured a partial transformation from corolla to calyx (Fig. S4i), or showed a complete transformation of androecium to gynoecium (Fig. S4j), or showed complete transformation of both corolla and androecium to the calyx and gynoecium, respectively (Fig. S4k). In the co-VIGS mutants that did not show homeotic variation of androecium to gynoecium, the pollen maturation of some flowers was impaired (Fig. S4l–n). qRT-PCR confirmed that the

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Fig. 3 Co-VIGS analyses of both PFDEF and PFTM6. a The wildtype (WT) flower. b, c Grade m1 of phenotypic variation. Part of corolla and androecia was transformed into calyx and gynoecia, respectively. d, e Grade m2 of phenotypic variation. Part of corolla was transformed into calyx, and all androecia were transformed into gynoecium. f–i Grade m3 of phenotypic variation. Corolla and androecium were transformed into calyx and gynoecium, respectively. Occasionally, the brown spot that was characteristic of corolla was still seen at the base of the transformed calyx (highlighted in a red arrow), and the gynoecia (indicated by white arrows) were separated in f, g. In some flowers, the gynoecia (indicated by white arrows) were fused in h, i. Part of calyx was removed to show the transformed calyx. j The relative PFDEF expression in the PFDEF– PFTM6-VIGS flowers with different grades (Wt0, m1, m2 and m3) of

phenotypic variations in comparison with that of wild type (WT). Wt0, WT-like flowers in the VIGS plants. k The PFTM6 expression in the PFDEF–PFTM6-VIGS flowers relative to WT. PFACTIN was used as an internal control. Gene amplification was repeated three times using the total RNA from each categorized flowers of the indicated plants (P1–P6). The mean and standard deviation are shown. l, m I2–KI staining of the wild-type pollen (l) and PFDEF–PFTM6VIGS pollen (m). The yellow pollen was immature and the blue pollen was mature. n Quantification of mature pollen rate in the WT and three PFDEF–PFTM6-VIGS flowers (#1–#3). o The PFDEF and PFTM6 expression in the androecia of flowers in n. Gene amplification was repeated three times using the total RNA from each flower as indicated. The mean and standard deviation are shown. Bars 2 mm (a–i), 100 lm (l, m)

PFDEF expression was more than 75 % of the WT while the PFGLO2 expression remained less than 50 % of the WT with extreme cases showing 10 % expression of the WT (Fig. S4o). Unlike the previous observation in the PFGLO1– PFGLO2-VIGS flowers (Zhang et al. 2014a), organ

number variation (Fig. S5) and obvious tip-bending variation of the transformed calyx were not observed in the double-gene downregulating mutants generated in the present work. Nevertheless, the large scale of VIGS analyses of both single and double genes (Table S1) suggested that PFGLO1 and PFDEF are essential to organ identity of

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Fig. 4 Functional analyses of the duplicated GLO–DEF gene pairs. PFGLO1–PFDEF-VIGS analyses. a A WT flower. b, c Grade m1 of phenotypic variation. Part of corolla and androecia was transformed into calyx and gynoecia, respectively. d, e Grade m2 of phenotypic variation. Part of corolla was transformed into calyx, and all androecia were transformed into gynoecia. f, g Grade m3 of phenotypic variation. Corolla and androecium were transformed into calyx and androecia, respectively. h The PFGLO1 expression in the PFGLO1–PFDEF-VIGS flowers. i The PFDEF expression in the PFGLO1–PFDEF-VIGS flowers. PFACTIN was used as an internal control. Gene amplification was repeated three times using the total RNA from each flower as from the indicated plants (P1–P6). The

mean and standard deviation are shown. PFGLO2–PFTM6-VIGS analyses. j, k The PFGLO2–PFTM6-VIGS flowers. l, m I2–KI staining of the wild-type pollen (l) and PFGLO2–PFTM6-VIGS pollen (m). The yellow pollen was immature and the blue pollen was mature. n Quantification of mature pollen rate in the WT and four PFGLO2–PFTM6-VIGS flowers (#1–#4). o The PFGLO2 and PFTM6 expression in the androecia of flowers in n. PFACTIN was used as an internal control. Gene amplification was repeated three times using the total RNA from each flower as indicated. The mean and standard deviation are shown. Bars 2 mm (a–g, j, k), 100 lm (l, m)

corolla and androecia, while PFGLO2 and PFTM6 are integrated into the developmental pathways for pollen maturation.

or/and PFDEF mutants was included. The pollen of 30–97 flowers from each genotype was examined using I2–KI staining. Compared to the wild type, only the severely downregulated PFGLO2- or/and PFTM6-associated mutants significantly developed flowers with reduced mature pollen (Fig. 5a). The variation of pollen maturation in the downregulated mutants of these Physalis B-class MADS-box genes ranged from 7.0 to 50.8 %. However, downregulation of PFGLO1 or/and PFDEF to the extent that did not lead to the homeotic mutation of transformation of androecium into gynoecium did not affect pollen maturation (Fig. 5b). These observations further supported that downregulating PFGLO2 or/and PFTM6 could affect pollen maturation. The fertility of the pollen in these

Roles of Physalis B-class genes in functionality of reproductive organs To further understand the role of these B-class genes in male fertility, we evaluated the pollen fertility. We made a statistical analysis on the frequency of flowers that developed immature pollen in WT plants and VIGS mutants. Since severe downregulation of PFGLO1 or PFDEF could change the organ identity of the androecium, no pollen was produced. Therefore, the weak downregulation of PFGLO1

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Fig. 5 Roles of Physalis B-class MADS-box genes in fertility. a Variation of pollen in the wild-type (WT) and the VIGS plants of B-class MADS-box genes. The ratio above each column indicates that rate to find the flowers with significant abnormal pollen. b Variation of mature pollen rate in three flowers of each case. The mean and the standard deviation are shown. c Fruit setting rate when the pollen from the indicated VIGS flowers was crossed to the WT stigmas. d Fruit setting rate when the WT pollen was crossed to the mutated stigmas. The numbers above each column in c and d indicate the fruit setting number in total crosses of each combination. The

crosses of the WT pollen and WT stigmas were used as controls, and 40 flowers were crossed for each combination in both c and d. The severely downregulated stigmas were used in d. Since downregulating PFGLO1 or PFDEF to less than 50 % of WT led to homeotic variation of androecia to gynoecia, the flowers that around 20–40 % of PFGLO1 or/and PFDEF was downregulated was included. In d, the fruit setting rate of the weak downregulated PFGLO1 or/and PFDEF flowers was indicated in black triangle. In all cases, PFGLO2 and PFTM6 were downregulated to less than 30 % of WT

B-class MADS-box genes knockdowns was further determined by crossing the pollen from the VIGS flowers with wild-type stigmas. These crosses did not result in a significant deviation in the fruit set as compared to the pollen– stigma crosses of the control (Fig. 5c). Moreover, the variation of the berry weight and the seed number per berry from the crosses of the mutated-pollen and the wild-type stigmas were similar to the control (Fig. S6). The flowers from the plants infected with empty TRV vectors (control) did not show any defects in pollen maturation (Fig. S7).

Thus, the downregulation of PFGLO2 or/and PFTM6 did not completely abolish the sterility of the androecium. The role of B-class MADS-box genes in female functionality was determined by crossing the wild-type pollen with the VIGS-mutated stigmas of these genes. Again, two types of gynoecia were investigated in PFGLO1- or/and PFDEF-associated mutants: one with the weak gene downregulation wherein the androecium identity was not altered and the gynoecia from the mutants wherein the androecium was completely transformed to gynoecium. In

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the first category with a weakly downregulated PFGLO1 or/and PFDEF, the rate of fruit set was similar to that of the WT control. Berries were not produced in the second category where PFGLO1 or/and PFDEF were strongly downregulated. However, fruit set in the PFGLO2-, PFTM6- and PFGLO2–PFTM6-VIGS flowers was comparable to the wild-type control (Fig. 5d). Moreover, in all cases that produced berries, the variation in berry weight and seed number per berry remained unchanged as compared to wild-type control (Fig. S8). Thus, PFGLO1 and PFDEF might have acquired an essential role in the functionality of gynoecia. In all these gene-silenced mutants, the pronounced variation in morphology (shape and size) of floral/fruiting calyx and carpel/fruit was not observed. Protein–protein interactions associated with B-class MADS-domain proteins Functional divergence might be partially due to the variation in protein subcellular localization and protein– protein interaction, as shown by the localization of PFGLO1 in the nuclei when PFDEF is present and PFGLO2 localizes in both nuclei and cytoplasm (Zhang et al. 2014a). We further analyzed the subcellular localization of PFDEF and PFTM6 to understand the underlying molecular basis of this functional divergence. Similar to the expression of GFP alone, expression of each GFP-fused construct of the two MADS-box genes in the plant cells resulted in a GFP signal in both nuclei and cytoplasm (Fig. 6a) suggesting that both PFDEF and PFTM6 may be localized in the two subcellular compartments. The physical interactions associated with these B-class MADS-domain proteins were further investigated via BiFC assays (Fig. 6b). In these assays, the yellow florescence protein was split into two halves: the N-terminal half (YFPn) and the C-terminal half (YFPc). Each of the two proteins was fused to each half of YFP. The two resulting constructs were co-expressed in plant cells, and the detection of YFP signal indicated the interaction of proteins and vice versa. This principle was used to investigate the dimerizations among B-class MADS-domain proteins in plant cells. Only PFGLO1 was found to form homodimers. Heterodimers PFGLO1–PFDEF and PFGLO2– PFTM6 were robustly formed, and no other heterodimerization were observed. Interestingly, homodimerization of PFGLO1 was observed only in the cytoplasm while the detected heterodimerizations occurred in the nuclei (highlighted in red box in Fig. 6b). The interactions of PFDEF and PFTM6 with known MADS-domain proteins MPF2, MPF3, PFAG, PFSEP1 and PFSEP3 in Physalis (He et al. 2007) were also evaluated in plant cells. PFTM6 interacted

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with MPF2 and MPF3, and PFGLO1 interacted with PFAG. The Physalis B-class MADS-domain proteins did not heterodimerize with PFSEP1 and PFSEP3 in plant cells and no other examples were detected in the BiFC system (Fig. 6b). The YFP signal for all the detected interactions in these protein–protein combinations was observed in the nuclei, indicating that the interaction facilitates the nuclei import of the B-class MADS-domain proteins. The interacting proteins associated with B-class MADS-domain proteins were characterized previously using yeast twohybrid assays (Zhang et al. 2014a). A comparison of the observations in plant cells and in yeast cells revealed that there were substantial discrepancies between the results of yeast two-hybrid and BiFC assays (Fig. 6c). This can be attributed to the weak and transient protein–protein interactions in the yeast two-hybrid system, which could be abolished by the potential existence of other partners that showed an extremely high affinity with either of the two proteins investigated in plant cells. Moreover, the high fidelity was not always observed in the interactions in yeast two-hybrid assays, which were also prone to artifacts. These assumptions for the inconsistency in the protein– protein interactions in the two distinct systems need further verifications; nonetheless, the consistency suggested that the B-class MADS-box proteins had different MADSdomain protein partners and patterns. Variations of gene expression in the downregulated mutants of B-class genes To further understand the functional divergence of these B-class MADS-box genes, we comprehensively analyzed the expression of some genes that are essential to confer the developmental role of these B-class genes. Cross-regulations among B-class MADS-box genes were first envisioned. The downregulation of PFGLO1, or/and PFDEF also downregulated the expression of PFGLO2, but PFTM6 was upregulated. However, the downregulation of PFGLO2, or/and PFTM6 did not alter the expression of PFGLO1 and PFDEF (Fig. 7a, b; Fig. S9), suggesting that PFGLO2 and PFTM6 were downstream targets of PFGLO1 and PFDEF. Downregulation of PFGLO1 reduced the PFDEF expression (Zhang et al. 2014a), and vice versa (Fig. S9) suggesting cross-activation of PFGLO1 and PFDEF. Moreover, downregulation of PFGLO2 and PFTM6 did not alter the expression of the other suggesting no cross-regulation between PFGLO2 and PFTM6. The expression of other genes was also investigated. Both MPF3 (an A-function MADS-box gene) and PFAG (a C-function MADS-box gene) genes (Zhao et al. 2013) were upregulated when PFGLO1 or/and PFDEF were downregulated. However, all these genes were not altered in the

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Fig. 6 Transient expression of B-class MADS-domain proteins in plant cells. a Subcellular localization of PFDEF and PFTM6. Three subsections (from left to right) are GFP alone, PFDEF–GFP and PFTM6–GFP, respectively, cloned in the Super 1300 vector. For each subsection, the GFP signal, bright background and merged image are shown from left to right. The arrow indicates the nuclei. b Protein– protein interaction in plant cells revealed by BiFC analyses. Yellow

signal indicates interaction of the two proteins investigated. The arrow indicates the nuclei. c Comparison of protein–protein interactions between B-class and other known Physalis MADS-domain proteins in BiFC/yeast two-hybrid assays. The interactions in yeast were summarized from Zhang et al. (2014a). A minus (-) indicates no interaction, a plus (?) indicates interaction, and the symbol ‘‘na’’ is abbreviated from the ‘‘not analyzed’’. Bars 20 lm (a, b)

PFGLO2 or PFTM6 downregulated mutated flowers (Fig. 7a, b; Fig. S9; Zhang et al. 2014a). In comparison with WT, MPF2 (He and Saedler 2005) was downregulated in all PFGLO1- or/and PFDEF-related downregulated flowers but not altered in the flowers that PFGLO2 or/and PFTM6 was downregulated (Fig. 7c). PFINV4 putatively encodes the invertase 4 (Zhao et al. 2013), which is a key gene implicated in sugar translocation pathway for pollen maturation (Oliver et al. 2005). PFINV4 was up-regulated in the PFGLO1- and PFDEF-related downregulated mutants while its expression remained unchanged in PFGLO2 or/and PFTM6 downregulated mutants (Fig. 7d).

These observations hinted that the role of PFGLO2 and PFTM6 in pollen maturation might be independent of the MPF3–MPF2/(–PFINV4) pathway. Therefore, the two obligate heterodimers PFGLO1– PFDEF and PFGLO2–PFTM6 evolved distinct regulatory networks.

Discussion Flowering plants evolved the GLO and DEF lineages of B-class MADS-box genes for organ identity specification

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Fig. 7 Gene expression variation in the B-class downregulated mutants. a The expression of MADS-box genes indicated in the PFGLO1–PFDEF-VIGS flowers. b The expression of MADS-box genes indicated in the PFGLO2–PFTM6-VIGS flowers. Flowers of different mutated grades (m1, m2 and m3) from three plants as indicated were investigated in each VIGS case. The column indicates the target gene for VIGS, and the gene name is highlighted in red. Other genes were indicated in each graph. c The expression of MPF2

in the downregulated mutants of B-class MADS-box genes. Three flowers for each case were checked. d The expression of PFINV4 in the downregulated mutants of B-class MADS-box genes. 3–6 Flowers in each case were checked. In these qRT-PCR analyses, PFACTIN was used as an internal control. Gene amplification was repeated three times using the total RNA from each flower as indicated. The mean and standard deviation are shown

and development of the corolla and androecium. In P. floridana, four functional B-class MADS-box genes are present; and the functional divergence of the two GLO-like genes PFGLO1 and PFGLO2 was revealed through deciphering the double-layered lantern mutant1 (Zhang et al. 2014a). In the present work, we used the TRV-mediated gene silencing approach to reveal the functions of PFDEF and PFTM6 in the DEF lineage, and the genetic interactions between all these B-class MADS-box genes in flower. The detailed studies of the resultant morphologies together

with the revealed divergence of the regulatory and interaction networks of these Physalis genes shed light on functional divergence of B-class MADS-box genes in angiosperms.

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Functional diversification of B-class MADS-box genes in Physalis floridana The robust and primary role of the B-class MADS-box genes is to specify the organ identity of corolla and

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androecium (Jack et al. 1992; Tro¨bner et al. 1992). This role was adapted by PFGLO1 and PFDEF in P. floridana since the severe downregulation of either PFGLO1 or/and PFDEF resulted in a complete homeotic variation of floral organs specified B-function genes. However, these genes functioned in the second and third floral whorls outside of organ identity specification. PFGLO1 (Zhang et al. 2014a) and PFDEF were also expressed in the primordia of gynoecium. Gene silencing did not result in abnormality of the gynoecium morphology but generated more gynoecia. Moreover, when we crossed the wild-type pollen with these stigmas, no berry was produced indicating poor functionality of these carpels. Thus, both PFGLO1 and PFDEF might have acquired a new role in the determination of functionality of the gynoecia. Silencing of these genes may affect the structure of the gynoecia or the pollen–stigma interaction, thus blocking fertilization process. This assumption needs verification. In this work, we observed that PFGLO1 did interact with PFAG, a putative C-function MADS-domain protein, but PFDEF did not. Nonetheless, downregulation of PFGLO1 or/and PFDEF resulted in the upregulation of PFAG. Furthermore, PFTM6 was also highly expressed in the gynoecium. These observations provide a possible link for these B-genes with development and functionality of gynoecium that has not been previously reported. This new role of these B-class genes in the functionality of gynoecium might be direct or indirect, and the molecular mechanism underlying this neofunctionalization requires further investigation. PFGLO2 and PFTM6 exert their role in male fertility and are essential for pollen maturation. MPF3 and MPF2 are essential for pollen development (He and Saedler 2005; Zhao et al. 2013); however, their expression was not altered in PFGLO2 or PFTM6 downregulated mutants. Moreover, the expression of PFINV4, a putative key gene in pollen maturation (Zhao et al. 2013), was not altered in the downregulated mutants of these B-class MADS-box genes. Therefore, the role of PFGLO2 and PFTM6 in pollen maturation might be independent of MPF3–MPF2/ (–PFINV4) associated pathways proposed previously (Zhao et al. 2013). Although, PFTM6 interacted with MPF2, pollen maturation was not completely abolished. Therefore, PFGLO2- or PFTM6-associated regulatory pathway might be largely parallel to other unidentified pollen developmental pathways in Physalis. The role of PFGLO1 or/and PFDEF in pollen maturation might be masked by their role in organ identity, since the organ identity specification is an earlier developmental stage than pollen maturation. However, PFINV4 was upregulated in the PFGLO1- and PFDEF-associated downregulated mutants, hinting that these two genes might act upstream of MPF3–MPF2/(–PFINV4) pathways with a hidden role in pollen development, since the MPF3–MPF2 regulatory

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circuit might activate the PFINV4 expression (Zhao et al. 2013). A direct repression of PFINV4 by PFGLO1 and PFDEF could not be excluded thus far. Hidden variability of the B-class MADS-box genes could provide a molecular basis for the evolution of novel functions (Geuten and Irish 2010) that may be revealed by downregulation of their double genes. Unlike PFGLO1 and PFGLO2 double knockouts (Zhang et al. 2014a), organ number variation and tip-bending development of the second floral whorl were not observed in the remaining downregulated double mutants indicating that the regulatory role on organ number and tip development of this floral whorl is specific to GLO genes in Physalis. In various plant species, functional divergence or altering gene expression could lead to diversified floral morphology (Kanno et al. 2003; Kramer et al. 2007; Mondrago´n-Palomino and Theissen 2011; Hofer et al. 2012; Sharma and Kramer 2013) and even affect fruit development (Yao et al. 2001; Poupin et al. 2007). The development of the ‘‘Chinese lantern’’ is associated with male fertility (He and Saedler 2005; Zhao et al. 2013) that is the primary role of B-class MADS-box genes. In P. floridana, the expression of most B-class genes is restricted to the corolla and androecium while PFTM6 is also relatively high expressed in calyx and carpels. Nonetheless, altering the expression of this gene did not lead to visible defects in the calyx and fruit development suggesting that interactions with other genes might be required for this potentially new function of PFTM6. Therefore, B-function MADS-box genes play developmental roles in multiple floral whorl organs, and their duplicates in Physalis have undergone a divergent process during evolution. Functional evolution of the B-class MADS-box genes within Solanaceae Gene duplication, diversification and redeployment play an important role in flower development and evolution (Irish and Litt 2005; Moore and Purugganan 2005; Innan and Kondrashov 2010). The auto- and cross-regulatory circuit of PI and AP3 in Arabidopsis and GLO and DEF Antirrhinum is required for the specification of corolla and androecium identity in angiosperms (Schwarz-Sommer et al. 1992; Zahn et al. 2005). The formation of specific multimeric MADS-domain protein complexes is essential in this process (Davies et al. 1996; Theißen and Saedler 2001; Lange et al. 2013). The duplicated B-class genes were lost in the basal asterids (Viaene et al. 2009) and the euasterids I (Lee and Irish 2011). However, the duplicates of both GLO and DEF lineages were maintained, which underwent substantial divergence within Solanaceae in flower development (Vandenbussche et al. 2004;

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Hernandez–Hernandez et al. 2007; Geuten and Irish 2010). Evolution of distinct molecular interactions among species defines the evolutionary trajectory of the duplicated paralogous genes (VanderSluis et al. 2010; Zhang et al. 2014a). The auto-regulatory and interacting relationship between the two B-class genes/proteins is quite simple in Arabidopsis and Antirrhinum; however, a complexity arises in Solanaceae owing to gene duplication. Nevertheless, the observed variations in molecular interactions did not completely explain the functional divergence patterns (Zhang et al. 2014a), suggesting a potential role of the coevolution of GLO/PI and DEF/AP3. The obligate heterodimerization of the GLO (PI)–DEF (AP3) evolved in angiosperms (Winter et al. 2002) preliminarily supporting this notion. Nevertheless, it needs further investigation. Functional evolution of the B-class genes was further discussed in two aspects. Evolution of lineage-specific paralogs The functional consequences of a mutation in any subunit duplicate of the obligate heterodimer pair support the hypothesis that both GLO1- and GLO2-like proteins (GLO-like) or DEF- and TM6-like proteins (DEF-like) exert their role as a functional unit to bear the complete B-function in some Solanaceous species, as the GLO or DEF proteins in Antirrhinum and PI or AP3 in Arabidopsis do. For each lineage, the two paralogous genes may have a synergistic role in a Solanaceous species. Albeit variability, the subfunctionalizational patterns of the duplicated B-class paralogous gene pair support the robustness and evolvability of the B-function genes (Geuten et al. 2011). Nonetheless, the two-lineage units seemed to show a separation with different divergent patterns along the phylogeny from Petunia to Physalis. A complete B-function is only present in PFGLO1 among all GLO-like members in Physalis (Zhang et al. 2014a), while the GLO duplicates in Petunia (Vandenbussche et al. 2004), Nicotiana (Geuten and Irish 2010) and Solanum (de Martino et al. 2006) are redundant with subfunctionalizations. The divergence of the DEF duplicates is quite different from the GLO duplicates. DEF-like fulfills a complete B-function in Nicotiana (Liu et al. 2004), Solanum (de Martino et al. 2006) and Physalis. The full B-function of DEF-like genes was shared by DEF (corolla) and TM6 (required for full function) in Petunia (Rijpkema et al. 2006). Single mutation of TM6-like genes in Petunia (Rijpkema et al. 2006) and in Physalis did not lead to any alterations in organ identity while TM6 mutations partially affected organ identity of androecium in Nicotiana (Geuten and Irish 2010) and Solanum (de Martino et al. 2006). PFTM6 in Physalis may have a potentially hidden role in the development of the calyx and fruit; however, it exerts its role in

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pollen maturation. The role of B-class MADS-box genes in anther development is supported by the observations outside of Solanaceae that the expression of AqAP3-1 is associated with the evolution of the sterile staminodium while silencing of AqAP3-2 primarily results in sterile stamens in Aquilegia; all stamens and staminodia in the doubly silenced flowers were transformed into carpels (Sharma and Kramer 2013). Evolution of obligate GLO–DEF heterodimer duplicates The complete B-function is associated with the evolution of the obligate GLO–DEF heterodimer mode (Tro¨bner et al. 1992; McGonigle et al. 1996; Winter et al. 2002; Zahn et al. 2005). Thus, the duplicated obligate GLO–DEF heterodimers demonstrated a functional separation in organ identity and organ functionality in Physalis. A similar functional divergence was observed in the duplicated C-class MADS-box genes in Antirrhinum where PLENA (PLE) plays a role in gynoecium identity while FARINELLI (FAR) accounts for male fertility (Davies et al. 1999). Variation of molecular interactions of B-class MADS-box genes/proteins contributes to the functional divergence. The formation of obligate GLO1–DEF and GLO2–TM6 during evolution accompanying the loss of other cross-heterodimerizations (Egea-Cortines et al. 1999; Leseberg et al. 2008; Immink et al. 2010; Zhang et al. 2014a) served as the key for functional separation of these duplicated heterodimer pair that occurred in both Solanum and Physalis. However, functional separation occurred in Physalis only. The distinct regulatory targets of the duplicated heterodimers (PFGLO1–PFDEF and PFGLO2– PFTM6) might also account for this divergence. In Physalis, PFGLO1 or/and PFDEF directly or indirectly regulate MPF3 (that controls calyx development; Zhao et al. 2013) and PFAG (that is mainly expressed in gynoecia). Moreover, only PFTM6 among these B-class MADS-box genes in Physalis is highly expressed in calyx and gynoecium indicating the potential role of these B-class genes in the development of calyx and gynoecium. Indeed, we observed the defects in functionality of gynoecium where PFGLO1 or/and PFDEF were severely downregulated suggesting that these B-class genes have acquired new developmental role. Therefore, duplication following subsequent divergence of B-class MADS-box genes may result in either functional diversification or/and floral morphological diversity (Irish and Litt 2005; Hernandez–Hernandez et al. 2007). A comprehensive comparison of molecular basis related to these B-class genes, as shown for the GLO-like paralogs in Physalis (Zhang et al. 2014a), and for AP3 and PI in Arabidopsis (Wuest et al. 2012) is required for complete

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understanding of the underlying molecular basis of B-function fulfillment and its diversifying patterns (subfunctionalization, redundancy and neofunctionalization). Nevertheless, the present findings in Physalis represent a new functionally divergent pattern of the duplicated B-class MADS-box genes within the Solanaceae, and will facilitate the understanding of the evolution of these genes in angiosperms. Author contributions CYH conceived and designed the experiments. SHZ and JSZ performed the in situ hybridizations and VIGS experiments. JZ performed subcellular localization and BiFC analyses. JSZ, SHZ, JZ and CYH analyzed the data. CYH wrote the manuscript. All authors have read and approved the manuscript. Acknowledgments This work was supported by the grants (31070203 and 91331103) from the National Natural Science Foundation of China. Conflict of interest of interest.

The authors declare that they have no conflict

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