Physalis floridana CRABS CLAW mediates neofunctionalization of GLOBOSA genes in carpel development.

Floral B-function MADS-box genes, such as GLOBOSA (GLO), function in corolla and stamen organ identity specification. The functions of these genes outside these floral whorls are rarely reported. DOLL1 is a GLO gene controlling corolla and androecium organ identity. In this study we found that, in Physalis floridana double-layered-lantern 1 (doll1) mutant pollinated with wild-type pollen, fruit set was extremely low, indicating that doll1 females are dysfunctional. Stigma and style structure, stigma receptivity, pollen tube guidance, and embryo sac development were also impaired in doll1. P. floridana CRABS CLAW (PFCRC), predominantly expressed in carpels, was repressed in doll1 native carpels. Loss-of-function of PFCRC altered carpel meristem determinacy, carpel closure, and ovule number, and the resultant 'pistil' consisted of multiple spirally-arranged dorsiventral carpels occasionally with 1-2 naked ovules on the margin and trichomes at each mutated carpel tip, implying an alteration of carpel organ identity. Regulatory and genetic interactions between B-class MADS-box genes and PFCRC were revealed in a context-dependent manner in floral development. Our work reveals a new role for the B-function genes in carpel and ovule development via regulating PFCRC, providing a new understanding of genetic regulatory networks between MADS-domain and CRC transcription factors in mediating carpel organ specification, functionality, and origin.

Characterization of the complete chloroplast genome of Pterocarya macroptera var. delavayi (Juglandaceae).

In this study, the complete chloroplast genome sequence of Pterocarya macroptera var. delavayi was reported and characterized. The chloroplast genome is 160,168 bp in length, and consists the typical quadripartite structure, a pair of inverted repeats (IRs, 26,007 bp) separated by a large single-copy region (89,701 bp) and a small single-copy region (18,453 bp). A total of 136 unique genes were predicted, including 88 protein-coding genes, 40 tRNA genes, and 8 rRNA genes. The GC content of the chloroplast genome is 36.2%. Phylogenetic analysis confirmed the close relationship between Pterocarya and Juglans.

Transcriptomic variation of the flower-fruit transition in Physalis and Solanum.

MAIN CONCLUSION: Gene expression variations in response to fertilization between Physalis and Solanum might play essential roles in species divergence and fruit evolution. Fertilization triggers variation in fruit development and morphology. The Chinese lantern, a morphological novelty derived from the calyx, is formed upon fertilization in Physalis but is not observed in Solanum. The underlying genetic variations are largely unknown. Here, we documented the developmental and morphological differences in the flower and fruit between Physalis floridana and Solanum pimpinellifolium and then evaluated both the transcript sequence variation and gene expression at the transcriptomic level at fertilization between the two species. In Physalis transcriptomic analysis, 468 unigenes were identified as differentially expressed genes (DEGs) that were strongly regulated by fertilization across 3 years. In comparison with tomato, 14,536 strict single-copy orthologous gene pairs were identified between P. floridana and S. pimpinellifolium in the flower-fruit transcriptome. Nine types of gene variations with specific GO-enriched patterns were identified, covering 58.82% orthologous gene pairs that were DEGs in either trend or dosage at the flower-fruit transition between the two species, which could adequately distinguish Solanum and Physalis, implying that differential gene expression at fertilization might play essential roles during the divergence and fruit evolution of Solanum-Physalis. Virus-induced gene silencing analyses revealed the developmental roles of some transcription factor genes in fertility, Chinese lantern development, and fruit weight control in Physalis. This study presents the first floral transcriptomic resource of Physalis, and reveals some candidate genetic variations accounting for the early fruit developmental evolution in Physalis in comparison to Solanum.

Distinct subfunctionalization and neofunctionalization of the B-class MADS-box genes in Physalis floridana.

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. 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 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.

Efficient gene silencing mediated by tobacco rattle virus in an emerging model plant physalis.

The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the 'Chinese lantern'). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS). Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution and development of the morphological diversities within the Solanaceae.

Deciphering the Physalis floridana double-layered-lantern1 mutant provides insights into functional divergence of the GLOBOSA duplicates within the Solanaceae.

Physalis spp. develop the "Chinese lantern" trait, also known as inflated calyx syndrome, that is a morphological novelty. Here, we identified the double-layered-lantern1 (doll1) mutant, a recessive and monofactorial mutation, in Physalis floridana; its corolla and androecium were transformed into the calyx and gynoecium, respectively. Two GLOBOSA-like MADS-box paralogous genes PFGLO1 and PFGLO2 were found in Physalis floridana, while the mutated phenotype was cosegregated with a large deletion harboring PFGLO1 and was complemented by the PFGLO1 genomic locus in transgenic plants, and severe PFGLO1 knockdowns phenocopied doll1. Thus, DOLL1 encodes the PFGLO1 protein and plays a primary role in determining corolla and androecium identity. However, specific PFGLO2 silencing showed no homeotic variation but rather affected pollen maturation. The two genes featured identical floral expression domains, but the encoding proteins shared 67% identity in sequences. PFGLO1 was localized in the nucleus when expressed in combination with a DEFICIENS homolog from Physalis floridana, whereas PFGLO2 was imported to the nucleus on its own. The two proteins were further found to have evolved different interacting partners and regulatory patterns, supporting the hypothesis that PFGLO2 is functionally separated from organ identity. Such a divergent pattern of duplicated GLO genes is unusual within the Solanaceae. Moreover, the phenotypes of the PFGLO1PFGLO2 double silencing mutants suggested that PFGLO2, through genetically interacting with PFGLO1, also exerts a role in the control of organ number and tip development of the second floral whorl. Our results, therefore, shed new light on the functional evolution of the duplicated GLO genes.

The euAP1 protein MPF3 represses MPF2 to specify floral calyx identity and displays crucial roles in Chinese lantern development in Physalis.

The Chinese lantern phenotype or inflated calyx syndrome (ICS) is a postfloral morphological novelty in Physalis. Its origin is associated with the heterotopic expression of the MADS box gene 2 from Physalis floridana (MPF2) in floral organs, yet the process underlying its identity remains elusive. Here, we show that MPF3, which is expressed specifically in floral tissues, encodes a core eudicot APETALA1-like (euAP1) MADS-domain protein. MPF3 was primarily localized to the nucleus, and it interacted with MPF2 and some floral MADS-domain proteins to selectively bind the CC-A-rich-GG (CArG) boxes in the MPF2 promoter. Downregulating MPF3 resulted in a dramatic elevation in MPF2 in the calyces and androecium, leading to enlarged and leaf-like floral calyces; however, the postfloral lantern was smaller and deformed. Starch accumulation in pollen was blocked. MPF3 MPF2 double knockdowns showed normal floral calyces and more mature pollen than those found in plants in which either MPF3 or MPF2 was downregulated. Therefore, MPF3 specifies calyx identity and regulates ICS formation and male fertility through interactions with MPF2/MPF2. Furthermore, both genes were found to activate Physalis floridana invertase gene 4 homolog, which encodes an invertase cleaving Suc, a putative key gene in sugar partitioning. The novel role of the MPF3-MPF2 regulatory circuit in male fertility is integral to the origin of ICS. Our results shed light on the evolution and development of ICS in Physalis and on the functional evolution of euAP1s in angiosperms.

Divergences of MPF2-like MADS-domain proteins have an association with the evolution of the inflated calyx syndrome within Solanaceae.

The inflated calyx syndrome (ICS) is a post-floral novelty within Solanaceae. Previous work has shown that MPF2-like MADS-box genes have been recruited for the development and evolution of ICS through heterotopic expression from vegetative to floral organs. ICS seems to be a plesiomorphic trait in Physaleae, but it has been secondarily lost in some lineages during evolution. We hypothesized that molecular and functional divergences of MPF2-like proteins might play a role in the loss of ICS. In this study we analyzed the phylogeny, selection and various functions of MPF2-like proteins with respect to the evolution of ICS. Directional selection of MPF2-like orthologs toward evolution of ICS was detected. While auto-activation capacity between proteins varies in yeast, MPF2-like interaction with floral MADS-domain proteins is robustly detected, hence substantiating their integration into the floral developmental programs. Dimerization with A- (MPF3) and E-function (PFSEP1/3) proteins seems to be essential for ICS development within Solanaceae. Moreover, the occurrence of the enlarged sepals, reminiscent of ICS, and MPF2-like interactions with these specific partners were observed in transgenic Arabidopsis. The interaction spectrum relevant to ICS seems to be plesiomorphic, reinforcing the plesiomorphy of this trait. The inability of some MPF2-like to interact with either the A-function or any of the E-function partners characterized is correlated with the loss of ICS in the lineages that showed a MPF2-like expression in the calyx. Our findings suggest that, after recruitment of MPF2-like genes for floral development, diversification in their coding region due to directional selection leads to a modification of the MADS-domain protein interacting spectrum, which might serve as a constraint for the evolution of ICS within Solanaceae.

Interactions among proteins of floral MADS-box genes in basal eudicots: implications for evolution of the regulatory network for flower development.

Floral MADS-box genes encode transcription factors that play critical roles in the development and evolution of the flower. Proteins of floral MADS-box genes regulate the expression of their downstream genes by forming various homodimers/heterodimers and quaternary complexes. Interactions among proteins of floral MADS-box genes have been documented in several model species, yet the information accumulated so far is still not sufficient to draw a general picture of the evolution of the interactions. We have characterized 28 putative floral MADS-box genes from three representative basal eudicots (i.e., Euptelea pleiospermum, Akebia trifoliata, and Pachysandra terminalis) and investigated the protein-protein interactions (PPIs) among the proteins encoded by these genes using yeast two-hybrid assays. We found that, although the PPIs in basal eudicots are largely consistent with those in core eudicots and monocots, there are lineage-specific features that have not been observed elsewhere. We also reconstructed the evolutionary histories of the PPIs among members of seven MADS-box gene lineages (i.e., AP1, AP3, PI, AG, STK, AGL2, and AGL9) in angiosperms. We revealed that the PPIs were extremely conserved in nine (or 32.1%) of the 28 possible combinations, whereas considerable variations existed in seven (25.0%) of them; in the remaining 12 (or 42.9%) combinations, however, no interaction was observed. Notably, most of the PPIs required for the formation of quaternary complexes, as suggested by the "quartet model," were highly conserved. This suggested that the evolutionarily conservative PPIs may have played critical roles in the establishment of the basic structure (or architecture) of the flower and experienced coevolution to maintain their functions. The evolutionarily variable PPIs, however, seem to have played subsidiary roles in flower development and have contributed to the variation in floral traits.