Genome evolution of the ancient hexaploid Platanus × acerifolia (London planetree).

Whole-genome duplication (WGD; i.e., polyploidy) and chromosomal rearrangement (i.e., genome shuffling) significantly influence genome structure and organization. Many polyploids show extensive genome shuffling relative to their pre-WGD ancestors. No reference genome is currently available for Platanaceae (Proteales), one of the sister groups to the core eudicots. Moreover, Platanus × acerifolia (London planetree; Platanaceae) is a widely used street tree. Given the pivotal phylogenetic position of Platanus and its 2-y flowering transition, understanding its flowering-time regulatory mechanism has significant evolutionary implications; however, the impact of Platanus genome evolution on flowering-time genes remains unknown. Here, we assembled a high-quality, chromosome-level reference genome for P. × acerifolia using a phylogeny-based subgenome phasing method. Comparative genomic analyses revealed that P. × acerifolia (2n = 42) is an ancient hexaploid with three subgenomes resulting from two sequential WGD events; Platanus does not seem to share any WGD with other Proteales or with core eudicots. Each P. × acerifolia subgenome is highly similar in structure and content to the reconstructed pre-WGD ancestral eudicot genome without chromosomal rearrangements. The P. × acerifolia genome exhibits karyotypic stasis and gene sub-/neo-functionalization and lacks subgenome dominance. The copy number of flowering-time genes in P. × acerifolia has undergone an expansion compared to other noncore eudicots, mainly via the WGD events. Sub-/neo-functionalization of duplicated genes provided the genetic basis underlying the unique flowering-time regulation in P. × acerifolia. The P. × acerifolia reference genome will greatly expand understanding of the evolution of genome organization, genetic diversity, and flowering-time regulation in angiosperms.

Genome-Wide Analysis of the BBX Genes in Platanus × acerifolia and Their Relationship with Flowering and/or Dormancy.

The B-BOX (BBX) gene family is widely distributed in animals and plants and is involved in the regulation of their growth and development. In plants, BBX genes play important roles in hormone signaling, biotic and abiotic stress, light-regulated photomorphogenesis, flowering, shade response, and pigment accumulation. However, there has been no systematic analysis of the BBX family in Platanus × acerifolia. In this study, we identified 39 BBX genes from the P. × acerifolia genome, and used TBtools, MEGA, MEME, NCBI CCD, PLANTCARE and other tools for gene collinearity analysis, phylogenetic analysis, gene structure, conserved domain analysis, and promoter cis-element analysis, and used the qRT-PCR and transcriptome data for analyzing expression pattern of the PaBBX genes. Collinearity analysis indicated segmental duplication was the main driver of the BBX family in P. × acerifolia, and phylogenetic analysis showed that the PaBBX family was divided into five subfamilies: I, II, III, IV and V. Gene structure analysis showed that some PaBBX genes contained super-long introns that may regulate their own expression. Moreover, the promoter of PaBBX genes contained a significant number of cis-acting elements that are associated with plant growth and development, as well as hormone and stress responses. The qRT-PCR results and transcriptome data indicated that certain PaBBX genes exhibited tissue-specific and stage-specific expression patterns, suggesting that these genes may have distinct regulatory roles in P. × acerifolia growth and development. In addition, some PaBBX genes were regularly expressed during the annual growth of P. × acerifolia, corresponding to different stages of flower transition, dormancy, and bud break, indicating that these genes may be involved in the regulation of flowering and/or dormancy of P. × acerifolia. This article provided new ideas for the study of dormancy regulation and annual growth patterns in perennial deciduous plants.

Regulation of alternative splicing of PaFT and PaFDL1, the FT and FD homologs in Platanus acerifolia.

Alternative splicing (AS) selects different alternative splice sites and produces a variety of transcripts with different exon/intron combinations, which may result in multiple protein isoforms. The splicing signals include cis-elements and RNA structures; however, the mechanisms of AS regulation in plants have yet to be elucidated. Previous studies have shown that in Platanus acerifolia, the FLOWERING LOCUS T (FT) homolog PaFT has a unique and complex AS pattern, in which most of the splice forms of PaFT involve the first and/or second intron, and the FD homolog PaFDL1 produces two transcripts via AS, whereas the other FT homolog PaFTL is not regulated by AS. In this study, the regulatory mechanism of the AS of PaFT was demonstrated to be conserved in different plant species. To define the distribution of the AS regulatory signals, the intron-swap, site-directed mutagenesis of alternative splice sites, and deletion experiment were performed. For the PaFT gene, all the signals that regulate the AS of the first intron were located within this intron, while the usage of the first alternative splice site in the second intron was determined by the first intron. Meanwhile, the AS of PaFDL1 might be co-regulated by exons and the first intron. Additionally, the first alternative splice site and adjacent region in PaFT intron 1 might contain cis-elements and/or RNA structures that affect the use of the other sites. This study had provided a deeper insight into the distribution of AS signals in plants, namely the AS signals of different splice sites might exist in the intron where the sites were present, and might also be distributed in exons or other introns.

PaNAC089 is a membrane-tethered transcription factor (MTTF) that modulates flowering, chlorophyll breakdown and trichome initiation.

Flowering and senescence are essential developmental stages of green plants, which are governed by complex molecular regulatory networks. However, the connection between flowering regulation and senescence regulation in London plane tree (Platanus acerifolia ) remains unknown. In this study, we identified a gene PaNAC089 from London plane tree, which encodes a membrane-tethered transcription factor (MTTF) belonging to the NAC (NAM, ATAF1/2, CUC2) transcription factor family. We investigated the functions of PaNAC089 in the regulation of flowering and senescence through the analysis of expression profiles and transgenic phenotypes. Heterologous overexpression of ΔPaNAC089 delayed flowering and inhibited chlorophyll breakdown to produce dark green rosette leaves in Arabidopsis . In addition, the trichome density of rosette leaves was decreased in transgenic lines. In ΔPaNAC089 overexpression plants, a series of functional genes with inhibited expression were identified by quantitative real-time polymerase chain reaction (qRT-PCR), including genes that regulate flowering, chlorophyll decomposition, and trichome initiation. Furthermore, Δ PaNAC089 directly binds to the promoter of CONSTANS (CO ) and NON-YELLOWING2 (NYE2 ) in the yeast one-hybrid assay. Consistent with this, luciferase (LUC) transient expression assays also showed that Δ PaNAC089 could inhibit the activity of NYE2 . To summarise, our data suggests that PaNAC089 is an MTTF that modulates flowering, chlorophyll breakdown and trichome initiation.

Two FD homologs from London plane (Platanus acerifolia) are associated with floral initiation and flower morphology.

The flowering-time gene FD encodes a bZIP transcription factor that interacts with FLOWERING LOCUS T (FT) to induce flowering in Arabidopsis. Previous research has identified two FT homologs of Platanus acerifolia, PaFT and PaFTL, which each have different expression patterns and are involved in diverse developmental processes. However, it is not known whether such FT/FD complexes participate in the flowering processes in P. acerifolia. Therefore, we isolated two closely related FD homologs, PaFDL1 and PaFDL2, and investigated their functions through the analysis of expression profiles, transgenic phenotypes, their interactions with different FT proteins, and potential cis-regulatory elements in their promoters. The PaFDL genes were found to display their maximal expression levels during the stage of floral transition, and subsequent expression patterns were also seen to be related to inflorescence developmental stage. In addition, both PaFDL1 and PaFDL2 were found to be subject to post-transcriptional alternative splicing, each gene producing two transcript forms. Transgenic tobacco overexpressing each of the four resulting transcript types displayed accelerated floral initiation and produced abnormal flowers. The results suggested that the complete PaFDL proteins may interact with different PaFT/PaFTL proteins in order to fulfill both conservative and diverse functions in floral initiation and floral development.