Variable expression of cyanide detoxification and tolerance genes in cyanogenic and acyanogenic white clover (Trifolium repens).

PREMISE: ß-Cyanoalanine synthase (ß-CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN). These functions are critical for all plants because HCN is produced at low levels during basic metabolic processes, and especially for cyanogenic species, which release high levels of HCN following tissue damage. However, expression of ß-CAS and Aox genes has not been examined in cyanogenic species, nor compared between cyanogenic and acyanogenic genotypes within a species. METHODS: We used a natural polymorphism for cyanogenesis in white clover to examine ß-CAS and Aox gene expression in relation to cyanogenesis-associated HCN exposure. We identified all ß-CAS and Aox gene copies present in the genome, including members of the Aox1, Aox2a, and Aox2d subfamilies previously reported in legumes. Expression levels were compared between cyanogenic and acyanogenic genotypes and between damaged and undamaged leaf tissue. RESULTS: ß-CAS and Aox2a expression was differentially elevated in cyanogenic genotypes, and tissue damage was not required to induce this increased expression. Aox2d, in contrast, appeared to be upregulated as a generalized wounding response. CONCLUSIONS: These findings suggest a heightened constitutive role for HCN detoxification (via elevated ß-CAS expression) and HCN-toxicity mitigation (via elevated Aox2a expression) in plants that are capable of cyanogenesis. As such, freezing-induced cyanide autotoxicity is unlikely to be the primary selective factor in the evolution of climate-associated cyanogenesis clines.

Haplotype-Resolved, Chromosome-Level Assembly of White Clover (Trifolium repens L., Fabaceae).

White clover (Trifolium repens L.; Fabaceae) is an important forage and cover crop in agricultural pastures around the world and is increasingly used in evolutionary ecology and genetics to understand the genetic basis of adaptation. Historically, improvements in white clover breeding practices and assessments of genetic variation in nature have been hampered by a lack of high-quality genomic resources for this species, owing in part to its high heterozygosity and allotetraploid hybrid origin. Here, we use PacBio HiFi and chromosome conformation capture (Omni-C) technologies to generate a chromosome-level, haplotype-resolved genome assembly for white clover totaling 998 Mbp (scaffold N50 = 59.3 Mbp) and 1 Gbp (scaffold N50 = 58.6 Mbp) for haplotypes 1 and 2, respectively, with each haplotype arranged into 16 chromosomes (8 per subgenome). We additionally provide a functionally annotated haploid mapping assembly (968 Mbp, scaffold N50 = 59.9 Mbp), which drastically improves on the existing reference assembly in both contiguity and assembly accuracy. We annotated 78,174 protein-coding genes, resulting in protein BUSCO completeness scores of 99.6% and 99.3% against the embryophyta_odb10 and fabales_odb10 lineage datasets, respectively.

Transcriptomic Analysis Suggests Auxin Regulation in Dorsal-Ventral Petal Asymmetry of Wild Progenitor Sinningia speciosa.

The establishment of dorsal-ventral (DV) petal asymmetry is accompanied by differential growth of DV petal size, shape, and color differences, which enhance ornamental values. Genes involved in flower symmetry in Sinningia speciosa have been identified as CYCLOIDEA (SsCYC), but which gene regulatory network (GRN) is associated with SsCYC to establish DV petal asymmetry is still unknown. To uncover the GRN of DV petal asymmetry, we identified 630 DV differentially expressed genes (DV-DEGs) from the RNA-Seq of dorsal and ventral petals in the wild progenitor, S. speciosa 'ES'. Validated by qRT-PCR, genes in the auxin signaling transduction pathway, SsCYC, and a major regulator of anthocyanin biosynthesis were upregulated in dorsal petals. These genes correlated with a higher endogenous auxin level in dorsal petals, with longer tube length growth through cell expansion and a purple dorsal color. Over-expression of SsCYC in Nicotiana reduced petal size by regulating cell growth, suggesting that SsCYC also controls cell expansion. This suggests that auxin and SsCYC both regulate DV petal asymmetry. Transiently over-expressed SsCYC, however, could not activate most major auxin signaling genes, suggesting that SsCYC may not trigger auxin regulation. Whether auxin can activate SsCYC or whether they act independently to regulate DV petal asymmetry remains to be explored in the future.

Plastome phylogenomics of Allaeanthus, Broussonetia and Malaisia (Dorstenieae, Moraceae) and the origin of B. × kazinoki.

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kozo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kozo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121-162,594 bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kozo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kozo appears to have a single origin and the possibility of a Korean origin cannot be ruled out.

Genetic Analysis of Floral Symmetry Transition in African Violet Suggests the Involvement of Trans-acting Factor for CYCLOIDEA Expression Shifts.

With the growing demand for its ornamental uses, the African violet (Saintpaulia ionantha) has been popular owing to its variations in color, shape and its rapid responses to artificial selection. Wild type African violet (WT) is characterized by flowers with bilateral symmetry yet reversals showing radially symmetrical flowers such as dorsalized actinomorphic (DA) and ventralized actinomorphic (VA) peloria are common. Genetic crosses among WT, DA, and VA revealed that these floral symmetry transitions are likely to be controlled by three alleles at a single locus in which the levels of dominance are in a hierarchical fashion. To investigate whether the floral symmetry gene was responsible for these reversals, orthologs of CYCLOIDEA (CYC) were isolated and their expressions correlated to floral symmetry transitions. Quantitative RT-PCR and in situ results indicated that dorsal-specific CYCs expression in WT S. ionantha (SiCYC and SiCYC1B) shifted in DA with a heterotopically extended expression to all petals, but in VA, SiCYC1s' dorsally specific expressions were greatly reduced. Selection signature analysis revealed that the major high-expressed copy of SiCYC had been constrained under purifying selection, whereas the low-expressed helper SiCYC1B appeared to be relaxed under purifying selection after the duplication into SiCYC and SiCYC1B. Heterologous expression of SiCYC in Arabdiopsis showed petal growth retardation which was attributed to limited cell proliferation. While expression shifts of SiCYC and SiCYC1B correlate perfectly to the resulting symmetry phenotype transitions in F1s of WT and DA, there is no certain allelic combination of inherited SiCYC1s associated with specific symmetry phenotypes. This floral transition indicates that although the expression shifts of SiCYC/1B are responsible for the two contrasting actinomorphic reversals in African violet, they are likely to be controlled by upstream trans-acting factors or epigenetic regulations.

Molecular recircumscription of Broussonetia (Moraceae) and the identity and taxonomic status of B. kaempferi var. australis.

BACKGROUND: Despite being a relatively small genus, the taxonomy of the paper mulberry genus Broussonetia remains problematic. Much of the controversy is related to the identity and taxonomic status of Broussonetia kaempferi var. australis, a name treated as a synonym in the floras of Taiwan and yet accepted in the floras of China. At the generic level, the monophyly of Corner (Gard Bull Singap 19:187-252, 1962)'s concept of Broussonetia has not been tested. In recent studies of Broussonetia of Japan, lectotypes of the genus were designated and three species (B. kaempferi, Broussonetia monoica, and Broussonetia papyrifera) and a hybrid (B. ×kazinoki) were recognized. Based on the revision and molecular phylogenetic analyses, this article aims to clarify these issues. RESULTS: Herbarium studies, field work, and molecular phylogenetic analyses indicate that all Taiwanese materials identifiable to B. kaempferi var. australis are conspecific with B. monoica of Japan and China. Molecular phylogenetic analyses showed that Broussonetia sensu Corner (Gard Bull Singap 19:187-252, 1962) contains two clades corresponding to sect. Broussonetia and sect. Allaeanthus, with Malaisia scandens sister to sect. Broussonetia. CONCLUSIONS: Based on our analyses, B. kaempferi var. australis is treated as a synonym of B. monoica and that B. kaempferi is not distributed in Taiwan. To correct the non-monophyly of Broussonetia sensu Corner (Gard Bull Singap 19:187-252, 1962), Broussonetia is recircumscribed to contain only sect. Broussonetia and the generic status of Allaeanthus is reinstated.