Functional importance and divergence of plant apurinic/apyrimidinic endonucleases in somatic and meiotic DNA repair.

Apurinic/apyrimidinic (AP) sites are one of the most abundant DNA lesions and are mainly repaired by AP endonucleases (APEs). While most eukaryotic genomes encode two APEs, plants usually possess three APEs, namely APE1L, APE2, and ARP. To date, the biological relevance and functional divergence of plant APEs are unclear. Here, we show that the three plant APEs have ancient origins, with the APE1L clade being plant-specific. In Arabidopsis thaliana, simultaneously mutating APE1L and APE2, but not ARP alone or in combination with either APE1L or APE2, results in clear developmental defects linked to genotoxic stress. Genetic analyses indicated that the three plant APEs have different substrate preferences in vivo. ARP is mainly responsible for AP site repair, while APE1L and APE2 prefer to repair 3'-blocked single-stranded DNA breaks. We further determined that APEs play an important role in DNA repair and the maintenance of genomic integrity in meiotic cells. The ape1l ape2 double mutant exhibited a greatly enhanced frequency of sporulation 1 (SPO11-1)-dependent and SPO11-1-independent double-stranded DNA breaks. The DNA damage response (DDR) was activated in ape1l ape2 to trigger pollen abortion. Our findings suggest functional divergence of plant APEs and reveal important roles of plant APEs during vegetative and reproductive development.

Co-option of a carotenoid cleavage dioxygenase gene (CCD4a) into the floral symmetry gene regulatory network contributes to the polymorphic floral shape-color combinations in Chrysanthemum sensu lato.

Morphological novelties, including formation of trait combinations, may result from de novo gene origination and/or co-option of existing genes into other developmental contexts. A variety of shape-color combinations of capitular florets occur in Chrysanthemum and its allies. We hypothesized that co-option of a carotenoid cleavage dioxygenase gene into the floral symmetry gene network would generate a white zygomorphic ray floret. We tested this hypothesis in an evolutionary context using species in Chrysanthemum sensu lato, a monophyletic group with diverse floral shape-color combinations, based on morphological investigation, interspecific crossing, molecular interaction and transgenic experiments. Our results showed that white color was significantly associated with floret zygomorphy. Specific expression of the carotenoid cleavage dioxygenase gene CCD4a in marginal florets resulted in white color. Crossing experiments between Chrysanthemum lavandulifolium and Ajania pacifica indicated that expression of CCD4a is trans-regulated. The floral symmetry regulator CYC2g can activate expression of CCD4a with a dependence on TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING (TCP) binding element 8 on the CCD4a promoter. Based on all experimental findings, we propose that gene co-option of carotenoid degradation into floral symmetry regulation, and the subsequent dysfunction or loss of either CYC2g or CCD4a, may have led to evolution of capitular shape-color patterning in Chrysanthemum sensu lato.

Rice Cell Division Cycle 20s are required for faithful chromosome segregation and cytokinesis during meiosis.

Chromosome segregation must be under strict regulation to maintain chromosome euploidy and stability. Cell Division Cycle 20 (CDC20) is an essential cell cycle regulator that promotes the metaphase-to-anaphase transition and functions in the spindle assembly checkpoint, a surveillance pathway that ensures the fidelity of chromosome segregation. Plant CDC20 genes are present in multiple copies, and whether CDC20s have the same functions in plants as in yeast and animals is unclear, given the potential for divergence or redundancy among the multiple copies. Here, we studied all three CDC20 genes in rice (Oryza sativa) and constructed two triple mutants by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated genome editing to explore their roles in development. Knocking out all three CDC20 genes led to total sterility but did not affect vegetative development. Loss of the three CDC20 proteins did not alter mitotic division but severely disrupted meiosis as a result of asynchronous and unequal chromosome segregation, chromosome lagging, and premature separation of chromatids. Immunofluorescence of tubulin revealed malformed meiotic spindles in microsporocytes of the triple mutants. Furthermore, cytokinesis of meiosis I was absent or abnormal, and cytokinesis II was completely prevented in all mutant microsporocytes; thus, no tetrads or pollen formed in either cdc20 triple mutant. Finally, the subcellular structures and functions of the tapetum were disturbed by the lack of CDC20 proteins. These findings demonstrate that the three rice CDC20s play redundant roles but are indispensable for faithful meiotic chromosome segregation and cytokinesis, which are required for the production of fertile microspores.

The Tetracentron genome provides insight into the early evolution of eudicots and the formation of vessel elements.

BACKGROUND: Tetracentron sinense is an endemic and endangered deciduous tree. It belongs to the Trochodendrales, one of four early diverging lineages of eudicots known for having vesselless secondary wood. Sequencing and resequencing of the T. sinense genome will help us understand eudicot evolution, the genetic basis of tracheary element development, and the genetic diversity of this relict species. RESULTS: Here, we report a chromosome-scale assembly of the T. sinense genome. We assemble the 1.07 Gb genome sequence into 24 chromosomes and annotate 32,690 protein-coding genes. Phylogenomic analyses verify that the Trochodendrales and core eudicots are sister lineages and showed that two whole-genome duplications occurred in the Trochodendrales approximately 82 and 59 million years ago. Synteny analyses suggest that the γ event, resulting in paleohexaploidy, may have only happened in core eudicots. Interestingly, we find that vessel elements are present in T. sinense, which has two orthologs of AtVND7, the master regulator of vessel formation. T. sinense also has several key genes regulated by or regulating TsVND7.2 and their regulatory relationship resembles that in Arabidopsis thaliana. Resequencing and population genomics reveals high levels of genetic diversity of T. sinense and identifies four refugia in China. CONCLUSIONS: The T. sinense genome provides a unique reference for inferring the early evolution of eudicots and the mechanisms underlying vessel element formation. Population genomics analysis of T. sinense reveals its genetic diversity and geographic structure with implications for conservation.

Insights into the Diversification and Evolution of R2R3-MYB Transcription Factors in Plants.

As one of the largest families of transcription factors (TFs) in plants, R2R3-MYB proteins play crucial roles in regulating a series of plant-specific biological processes. Although the diversity of plant R2R3-MYB TFs has been studied previously, the processes and mechanisms underlying the expansion of these proteins remain unclear. Here, we performed evolutionary analyses of plant R2R3-MYB TFs with dense coverage of streptophyte algae and embryophytes. Our analyses revealed that ancestral land plants exhibited 10 subfamilies of R2R3-MYB proteins, among which orthologs of seven subfamilies were present in chlorophytes and charophycean algae. We found that asymmetric gene duplication events in different subfamilies account for the expansion of R2R3-MYB proteins in embryophytes. We further discovered that the largest subfamily of R2R3-MYBs in land plants, subfamily VIII, emerged in the common ancestor of Zygnematophyceae and embryophytes. During plant terrestrialization, six duplication events gave rise to seven clades of subfamily VIII. Subsequently, this TF subfamily showed a tendency for expansion in bryophytes, lycophytes, and ferns and extensively diversified in ancestral gymnosperms and angiosperms in clades VIII-A-1, VIII-D, and VIII-E. In contrast to subfamily VIII, other subfamilies of R2R3-MYB TFs have remained less expanded across embryophytes. The findings regarding phylogenetic analyses, auxiliary motifs, and DNA-binding specificities provide insight into the evolutionary history of plant R2R3-MYB TFs and shed light on the mechanisms underlying the extensive expansion and subsequent sub- and neofunctionalization of these proteins.

To be serrate or pinnate: diverse leaf forms of yarrows (Achillea) are linked to differential expression patterns of NAM genes.

Background and Aims: To understand the link between species diversity and phenotype developmental evolution is an important issue in evolutionary biology. Yarrows in the genus Achillea (Asteraceae) show a great diversity in leaf serrate or pinnate dissection patterns. In Arabidopsis thaliana, the development of leaf serration requires the activity of the transcription factor CUC2. Does this regulator also work for leaf dissections of the Asteraceae plants? If so, how do the conserved regulatory 'tools' work differently to produce diverse leaf forms? Methods: Seedling leaf morphology was observed, and morphogenesis of leaf serration or lobes was examined by scanning electron microscopy (SEM). NAM genes, orthologues of arabidopsis CUC2, were isolated from A. acuminata with serrate leaves and A. asiatica with three-pinnatisect leaves, respectively. By means of whole-mount in situ mRNA hybridization and two quantitative gene expression assays, the droplet digital PCR (ddPCR) and quantitative real-time PCR (qPCR), expression patterns of the NAM genes during leaf dissection development were checked in both species for comparison. Key Results: For both species, the development of leaf dissection initiated when a leaf blade was about 300-400 µm long. In A. acuminata, in situ hybridization showed NAM expression signals at leaf margins where teeth are growing, or later on, in the sinuses of the teeth, whilst in A. asiatica, hybridization signals appear not only on leaf margins but further on the margins of leaf lobes. Both ddPCR and qPCR revealed a continuous decline of AacNAM expression from the early to the late developmental stages of a single leaf of A. acuminata, whereas a relatively long maintenance and fluctuation of AasNAM expression was seen in a leaf of A. asiatica. Conclusions: Differential spatiotemporal patterns of NAM expression were found between the two yarrow species during development of leaf dissection. This study provides the first evidence for NAM activity in the development of leaf dissection of the Asteraceae plants, and demonstrates that leaf form diversity is correlated to the altered NAM expression dynamic.

Evolutionary Analysis of the LAFL Genes Involved in the Land Plant Seed Maturation Program.

Seeds are one of the most significant innovations in the land plant lineage, critical to the diversification and adaptation of plants to terrestrial environments. From perspective of seed evo-devo, the most crucial developmental stage in this innovation is seed maturation, which includes accumulation of storage reserves, acquisition of desiccation tolerance, and induction of dormancy. Based on previous studies of seed development in the model plant Arabidopsis thaliana, seed maturation is mainly controlled by the LAFL regulatory network, which includes LEAFY COTYLEDON1 (LEC1) and LEC1-LIKE (L1L) of the NF-YB gene family, and ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEC2 (LEAFY COTYLEDON2) of the B3-AFL gene family. In the present study, molecular evolution of these LAFL genes was analyzed, using representative species from across the major plant lineages. Additionally, to elucidate the molecular mechanisms of the seed maturation program, co-expression pattern analyses of LAFL genes were conducted across vascular plants. The results show that the origin of AFL gene family dates back to a common ancestor of bryophytes and vascular plants, while LEC1-type genes are only found in vascular plants. LAFL genes of vascular plants likely specify their co-expression in two different developmental phrases, spore and seed maturation, respectively, and expression patterns vary slightly across the major vascular plants lineages. All the information presented in this study will provide insights into the origin and diversification of seed plants.