Mingai Li

Polyploidization has played a central role in the evolutionary history of most plant lineages, yet it poses significant challenges for phylogenetic inference, particularly in allopolyploid complexes with reticulate species relationships. Luzula sect. Luzula (Juncaceae) is a taxonomically intricate group characterized by widespread polyploidy, agmatoploidy, and high morphological uniformity. Focusing on the Eastern Alps, a key center of its diversity, we collected 1002 samples of nine species and applied an integrative framework combining ddRADseq, plastid sequencing, relative genome size estimation, and chromosome counting to disentangle its evolutionary history. We extended previously inferred phylogenetic relationships and assessed gene flow among diploids, establishing a baseline for investigating the origin of polyploids. By analyzing patterns of genotype frequencies and genetic affinities to diploids, we inferred the most likely parental species of polyploids and identified key hybridization events shaping the current taxonomic and karyotypic diversity within this group. Our results reveal weak genetic differentiation among some diploid lineages, likely reflecting gene flow and incomplete lineage sorting. We propose a common allopolyploid origin of two tetraploids, which subsequently gave rise to a third tetraploid and a hexaploid species through interploidy hybridization. Although the parental species of some polyploids remain obscure, our genomic data highlight polyploidy and hybridization as major drivers of speciation in this poorly understood lineage. This study underscores the value of integrative approaches in resolving reticulate plant phylogenies and advances our understanding of polyploid speciation.

The funarioid moss species Physcomitrium eurystomum, which is threatened with extinction, was the subject of this study. The riparian habitat type of this species is often under the influence of contaminated water, and, therefore, we tested the influence of selected potentially toxic elements (PTEs), namely zinc and copper, on the development, physiological features, and survival of the species on two different accessions (German and Croatian). The results obtained showed the different resilience of the two accessions to the PTEs. Flow cytometry analyses revealed that the two accessions differ significantly in terms of genome size. However, the different amplitude of resilience to the tested PTEs, the divergence in physiological responses, and survival within two accessions of the same species are confirmed, as well as the dissimilarity of their genome size, likely associated with ploidy level difference and possibly distinct hybrid origin.

Despite the paramount importance in metal(loid) detoxification by phytochelatin synthase (PCS) genes, no comprehensive analysis of their evolutionary patterns has been carried out in land plants in general and in crops in particular. A phylogenetic large-scale analysis of gene duplication in angiosperms was carried out followed by in vitro recombinant protein assays as well as complementation analysis (growth, thiol-peptides, elements) of Arabidopsis cad1-3 mutant with four representative PCS genes from two model crop species, Malus domestica and Medicago truncatula. We uncovered a so far undetected ancient tandem duplication (D duplication) spanning the whole core eudicotyledon radiation. Complementation with PCS genes from both D-subclades from M. domestica and M. truncatula displayed clear in vivo conservation of the differences between D1 and D2 paralogous proteins in plant growth, phytochelatin and glutathione pools, as well as element contents under heavy metal(loid) stress. In vitro recombinant PCS analysis identified analogous patterns of differentiation, showing a higher activity of D2 PCS genes, so far largely overlooked, compared to their paralogs from the D1 clade. This suggests that in many other crop species where the duplication is present, the D2 copy might play a significant role in metal(loid) detoxification. The retention of both PCS paralogs and of their functional features for such long divergence time suggests that PCS copy number could be constrained by functional specialization and/or gene dosage sensitivity. These results uncover the patterns of PCS evolution in plant genomes and of functional specialization of their paralogs in the genomes of two important model crops.

Plantago (plantains, Plantaginaceae) is a cosmopolitan genus including over 250 species used as functional foods, forage, and traditional medicine. Among them, Plantago lanceolata is commonly used as an ingredient of herbal products, but the close similarity to other Plantago species can cause misidentifications with potentially serious consequences for product safety/quality. To test the possibility of developing species-specific barcoding markers, we de novo assembled plastome sequences of individuals of Plantago argentea, Plantago atrata, P. lanceolata, and Plantago maritima. These genomes were characterized in comparison with both previously sequenced conspecific accessions and other publicly available plastomes, thus providing an assessment of both intraspecific and interspecific genetic variation in Plantago plastomes. Additionally, molecular evolutionary analyses indicated that eleven protein-coding genes involved in different plastid functions in Plantago plastomes underwent positive selection, suggesting they might have contributed to enhancing species’ adaptation during the evolutionary history of Plantago. While the most variable mutational hotspots in Plantago plastomes were not suitable for the development of species-specific molecular markers, species-specific polymorphisms could discriminate P. lanceolata from its closest relatives. Taken together, these results highlight the potential of plastome sequencing for the development of molecular markers to improve the identification of species with relevance in herbal products.

With the advent of genomic and other omics technologies, the last decades have witnessed a series of steady and important breakthroughs in the understanding of genetic determinants of different reproductive systems in vascular plants and especially on how sexual reproduction shaped their evolution. In contrast, the molecular mechanisms of these fundamental aspects of the biology of bryophytes, a group of non-vascular embryophyte plants sister to all tracheophytes, are still largely obscure. The recent characterization of the sex chromosomes and genetic switches determining sex in bryophytes and emerging approaches for molecular sexing of gametophytes hold great promise for elucidation of the evolutionary history as well as the conservation of this species-rich but understudied group of land plants.

The capacity to emit isoprene, among other stresses, protects plants from drought, but the molecular mechanisms underlying this trait are only partly understood. The Arecaceae (palms) constitute a very interesting model system to test the involvement of isoprene in enhancing drought tolerance, as their high isoprene emissions may have contributed to make them hyperdominant in neotropical dry forests, characterized by recurrent and extended periods of drought stress. In this study we isolated and functionally characterized a novel isoprene synthase, the gene responsible for isoprene biosynthesis, from Copernicia prunifera, a palm from seasonally dry tropical forests. When overexpressed in the non-emitter Arabidopsis thaliana, CprISPS conferred significant levels of isoprene emission, together with enhanced tolerance to water limitation throughout plant growth and development, from germination to maturity. CprISPS overexpressors displayed higher germination, cotyledon/leaf greening, water usage efficiency, and survival than WT Arabidopsis under various types of water limitation. This increased drought tolerance was accompanied by a marked transcriptional up-regulation of both ABA-dependent and ABA-independent key drought response genes. Taken together, these results demonstrate the capacity of CprISPS to enhance drought tolerance in Arabidopsis and suggest that isoprene emission could have evolved in Arecaceae as an adaptive mechanism against drought.

SUMMARY: Phloridzin is the most abundant polyphenolic compound in apple (Malus × domestica Borkh.), which results from the action of a key phloretin‐specific UDP‐2′‐O‐glucosyltransferase (MdPGT1). Here, we simultaneously assessed the effects of targeting MdPGT1 by conventional transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐mediated genome editing. To this end, we conducted transcriptomic and metabolic analyses of MdPGT1 RNA interference knockdown and genome‐edited lines. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology, whereas genome‐edited lines exhibited normal growth despite reduced foliar phloridzin. RNA‐sequencing analysis identified a common core of regulated genes, involved in phenylpropanoid and flavonoid pathways. However, we identified genes and processes differentially modulated in stunted and genome‐edited lines, including key transcription factors and genes involved in phytohormone signalling. Therefore, we conducted a phytohormone profiling to obtain insight into their role in the phenotypes observed. We found that salicylic and jasmonic acid were increased in dwarf lines, whereas auxin and ABA showed no correlation with the growth phenotype. Furthermore, bioactive brassinosteroids were commonly up‐regulated, whereas gibberellin GA₄ was distinctively altered, showing a sharp decrease in RNA interference knockdown lines. Expression analysis by reverse transcriptase‐quantitative polymerase chain reaction expression analysis further confirmed transcriptional regulation of key factors involved in brassinosteroid and gibberellin interaction. These findings suggest that a differential modulation of phytohormones may be involved in the contrasting effects on growth following phloridzin reduction. The present study also illustrates how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple.