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.

Exon junction complex (EJC) core genes play multiple developmental roles in Physalis floridana.

KEY MESSAGE: Molecular and functional characterization of four gene families of the Physalis exon junction complex (EJC) core improved our understanding of the evolution and function of EJC core genes in plants. The exon junction complex (EJC) plays significant roles in posttranscriptional regulation of genes in eukaryotes. However, its developmental roles in plants are poorly known. We characterized four EJC core genes from Physalis floridana that were named PFMAGO, PFY14, PFeIF4AIII and PFBTZ. They shared a similar phylogenetic topology and were expressed in all examined organs. PFMAGO, PFY14 and PFeIF4AIII were localized in both the nucleus and cytoplasm while PFBTZ was mainly localized in the cytoplasm. No protein homodimerization was observed, but they could form heterodimers excluding the PFY14-PFBTZ heterodimerization. Virus-induced gene silencing (VIGS) of PFMAGO or PFY14 aborted pollen development and resulted in low plant survival due to a leaf-blight-like phenotype in the shoot apex. Carpel functionality was also impaired in the PFY14 knockdowns, whereas pollen maturation was uniquely affected in PFBTZ-VIGS plants. Once PFeIF4AIII was strongly downregulated, plant survival was reduced via a decomposing root collar after flowering and Chinese lantern morphology was distorted. The expression of Physalis orthologous genes in the DYT1-TDF1-AMS-bHLH91 regulatory cascade that is associated with pollen maturation was significantly downregulated in PFMAGO-, PFY14- and PFBTZ-VIGS flowers. Intron-retention in the transcripts of P. floridana dysfunctional tapetum1 (PFDYT1) occurred in these mutated flowers. Additionally, the expression level of WRKY genes in defense-related pathways in the shoot apex of PFMAGO- or PFY14-VIGS plants and in the root collar of PFeIF4AIII-VIGS plants was significantly downregulated. Taken together, the Physalis EJC core genes play multiple roles including a conserved role in male fertility and newly discovered roles in Chinese lantern development, carpel functionality and defense-related processes. These data increase our understanding of the evolution and functions of EJC core genes in plants.

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.

MPF2-like-a MADS-box genes control the inflated Calyx syndrome in Withania (Solanaceae): roles of Darwinian selection.

The Chinese lantern, which is the inflated calyx syndrome (ICS) of Physalis, is formed by MPF2 in the presence of the plant hormones, cytokinin and gibberellin. MPF2 knockdown mutants of Physalis have small leaves, no ICS, and are male sterile, thus, revealing three MPF2-related functions. Of the close relatives of Physalis, Tubocapsicum has only a rudimentary calyx, whereas others, like the Withania species, have ICS. From all Withania samples tested, two classes of MPF2-like orthologs, MPF2-like-A and MPF2-like-B, were isolated, whereas only the latter class was obtained from tetraploid Tubocapsicum. Though distinct differences can be observed between MPF2-like-A and MPF2-like-B proteins, that is MPF2-like-A proteins have an aberrant structure in that they have a three amino acid deletion in their C-domain and an eight amino acid extension at the C-terminal end, MPF2-like-A genes are phylogenetically closer to the Physalis MPF2-like genes. Unlike MPF2-like-B, the overexpression of MPF2-like-A in Arabidopsis revealed extra large sepals thus suggesting that MPF2-like-A genes are very likely responsible for the ICS formation in Withania. This correlated with the expression pattern of MPF2-like-A in vegetative and flower tissues, whereas MPF2-like-B is expressed only in vegetative tissues of Withania. In Tubocapsicum, however, MPF2-like-B RNA is detectable in all tissues tested. Finally, positive Darwinian selection was observed in the branch leading to Physalis MPF2-like and Withania MPF2-like-A proteins, followed by purifying selection once the trait had evolved. By contrast, purifying selection was detected for all other MPF2-like proteins tested. The contribution of the MPF2-like gene duplication to subfunctionalization is discussed.

Hormonal control of the inflated calyx syndrome, a morphological novelty, in Physalis.

The 'Chinese lantern' phenotype or inflated calyx syndrome (ICS)--inflated sepals encapsulating the mature berry of Physalis floridana--is a morphological novelty within the Solanaceae. ICS is associated with heterotopic expression of MPF2, which codes for a MADS-box transcription factor otherwise involved in leaf formation and male fertility. In accordance with this finding, the MPF2 promoter sequence differs significantly from that of its orthologue STMADS16 in the related Solanum tuberosum, which does not exhibit ICS. However, heterotopic expression of MPF2 is not sufficient for ICS formation in P. floridana- fertilization is also important. Here we report that the hormones cytokinin and gibberellin are essential for ICS formation. MPF2 controls sepal cell division, but the resulting cells are small. Calyx size increases substantially only if gibberellin and cytokinin are available to promote cell elongation and further cell division. Transient expression of appropriate MPF2-/STMADS16-GFP fusions in leaf tissues in the presence of hormones revealed that cytokinin, but not gibberellin, facilitated transport of the transcription factor into the nucleus. Furthermore, an ICS-like structure can be induced in transgenic S. tuberosum by ectopic expression of STMADS16 and simultaneous treatment with cytokinin and gibberellin. Strikingly, transgenic Arabidopsis ectopically expressing solanaceous MPF2-like proteins display enhanced sepal growth when exposed to cytokinin only, while orthologous proteins from non-solanaceous plants did not require cytokinin for this function. These data are incorporated into a detailed model for ICS formation in P. floridana.