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Athyrium yokoscense is hypertolerant to cadmium (Cd) and can grow normally under a high Cd concentration despite Cd being a highly toxic heavy metal. To mitigate Cd stress in general plant species, Cd is promptly chelated with a thiol compound and is isolated into vacuoles. Generated active oxygen species (ROS) in the cytoplasm are removed by reduced glutathione. However, we found many differences in the countermeasures in A. yokoscense. Thiol compounds accumulated in the stele of the roots, although a long-term Cd exposure induced Cd accumulation in the aerial parts. Synchrotron radiation-based X-ray fluorescence (SR-XRF) analysis indicated that a large amount of Cd was localized in the cell walls of the roots. Overexpression of AyNramp5a, encoding a representative Fe and Mn transporter of A. yokoscense, increased both Cd uptake and Fe and Mn uptake in rice calli under the Cd exposure conditions. Organic acids are known to play a key role in reducing Cd availability to the plants by forming chelation and preventing its entry in free form into the roots. In A. yokoscense roots, Organic acids were abundantly detected. Investigating the chemical forms of the Cd molecules by X-ray absorption fine structure (XAFS) analysis detected many compounds with Cd-oxygen (Cd-O) binding in A. yokoscense roots, whereas in the aerial parts, the ratio of the compounds with Cd-sulfur (Cd-S) binding was increased. Together, our results imply that the strong Cd tolerance of A. yokoscense is an attribute of the following two mechanisms: Cd-O compound formation in the cell wall is a barrier to reduce Cd uptake into aerial parts. Thiol compounds in the region of root stele are involved in detoxication of Cd by formation of Cd-S compounds., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Cytoplasmic male sterility (CMS) is a mitochondrial-encoded trait that confers reproductive defects in males but not in females or any vegetative function. Why CMS is so often found in plants should be investigated from the viewpoint of mitochondrial phylogeny. Beta vulgaris, including the wild subspecies maritima and cultivated subspecies vulgaris (e.g., sugar beet), is known to be mitochondrially polymorphic, from which multiple CMS mitochondria have been found, but their evolutionary relationship has been obscure. We first refined the B. vulgaris reference mitochondrial genome to conduct a more accurate phylogenetic study. We identified mitochondrial single-nucleotide polymorphic sites from 600 B. vulgaris accessions. Principal component analysis, hierarchical clustering analysis, and creation of a phylogenetic tree consistently suggested that B. vulgaris mitochondria can be classified into several groups whose geographical distribution tends to be biased toward either the Atlantic or Mediterranean coasts. We examined the distribution of CMS-associated mitochondrial genes from Owen, E- and G-type CMS mitochondria. About one-third of cultivated beets had Owen-type CMS, which reflects the prevalence of using Owen-type CMS in hybrid breeding. Occurrence frequencies for each of the three CMS genes in wild beet were less than 4%. CMS genes were tightly associated with specific mitochondrial groups that are phylogenetically distinct, suggesting their independent origin. However, homologous sequences of the Owen type CMS gene occurred in several different mitochondrial groups, for which an intricate explanation is necessary. Whereas the origin of cultivated beet had been presumed to be Greece, we found an absence of Owen-type mitochondria in Greek accessions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kubota et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Heteroplasmy, the coexistence of multiple mitochondrial DNA (mtDNA) sequences in a cell, is well documented in plants. Next-generation sequencing technology (NGS) has made it feasible to sequence entire genomes. Thus, NGS has the potential to detect heteroplasmy; however, the methods and pitfalls in heteroplasmy detection have not been fully investigated and identified. One obstacle for heteroplasmy detection is the sequence homology between mitochondrial-, plastid-, and nuclear DNA, of which the influence of nuclear DNA segments homologous to mtDNA (numt) need to be minimized. To detect heteroplasmy, we first excluded nuclear DNA sequences of sugar beet (Beta vulgaris) line EL10 from the sugar beet mtDNA sequence. NGS reads were obtained from single plants of sugar beet lines NK-195BRmm-O and NK-291BRmm-O and mapped to the unexcluded mtDNA regions. More than 1000 sites exhibited intra-individual polymorphism as detected by genome browsing analysis. We focused on a 309-bp region where 12 intra-individual polymorphic sites were closely linked to each other. Although the existence of DNA molecules having variant alleles at the 12 sites was confirmed by PCR amplification from NK-195BRmm-O and NK-291BRmm-O, these variants were not always called by six variant-calling programs, suggesting that these programs are inappropriate for intra-individual polymorphism detection. When we changed the nuclear DNA reference, a numt absent from EL10 was found to include the 309-bp region. Genetic segregation of an F2 population from NK-195BRmm-O x NK-291BRmm-O supported the numt origin of the variant alleles. Using four references, we found that numt detection exhibited reference dependency, and extreme polymorphism of numts exists among sugar beet lines. One of the identified numts absent from EL10 is also associated with another intra-individual polymorphic site in NK-195mm-O. Our data suggest that polymorphism among numts is unexpectedly high within sugar beets, leading to confusion about the true degree of heteroplasmy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Taniguchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cytoplasmic male sterility (CMS) is an increasingly important issue within the context of hybrid seed production. Its genetic framework is simple: S-cytoplasm for male sterility induction and dominant allele of the restorer-of-fertility gene (Rf) for suppression of S. However, breeders sometimes encounter a phenotype of CMS plants too complex to be explained via this simple model. The molecular basis of CMS provides clue to the mechanisms that underlie the expression of CMS. Mitochondria have been associated with S, and several unique ORFs to S-mitochondria are thought to be responsible for the induction of male sterility in various crops. Their functions are still the subject of debate, but they have been hypothesized to emit elements that trigger sterility. Rf suppresses the action of S by various mechanisms. Some Rfs, including those that encode the pentatricopeptide repeat (PPR) protein and other proteins, are now considered members of unique gene families that are specific to certain lineages. Additionally, they are thought to be complex loci in which several genes in a haplotype simultaneously counteract an S-cytoplasm and differences in the suite of genes in a haplotype can lead to multiple allelism including strong and weak Rf at phenotypic level. The stability of CMS is influenced by factors such as the environment, cytoplasm, and genetic background; the interaction of these factors is also important. In contrast, unstable CMS becomes inducible CMS if its expression can be controlled. CMS becomes environmentally sensitive in a genotype-dependent manner, suggesting the feasibility of controlling the expression of CMS., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Cytoplasmic male sterility in sugar beet becomes thermo-sensitive when combined with specific genotypes, potentially offering a means to environmentally control pollination by this trait. The stability of cytoplasmic male sterility expression in several genetic backgrounds was investigated in sugar beet (Beta vulgaris L.). Nine genetically heterogenous plants from open-pollinated varieties were crossed with a cytoplasmic male sterile line to obtain 266 F 1 plants. Based on marker analysis using a multiallelic DNA marker linked to restorer-of-fertility 1 (Rf1), we divided the F 1 plants into 15 genotypes. We evaluated the phenotypes of the F 1 plants under two environmental conditions: greenhouse rooms with or without daytime heating during the flowering season. Three phenotypic groups appeared: those consistently expressing male sterility, those consistently having restored pollen fertility, and those expressing male sterility in a thermo-sensitive manner. All plants in the consistently male sterile group inherited a specific Rf1 marker type named p4. We tested the potential for thermo-sensitive male sterile plants to serve as seed parents for hybrid seed production, and three genotypes were selected. Open pollination by a pollen parental line with a dominant trait of red-pigmented hypocotyls and leaf veins resulted in seed setting on thermo-sensitive male sterile plants, indicating that their female organs were functional. More than 99.9% of the progeny expressed the red pigmentation trait; hence, highly pure hybrids were obtained. We determined the nucleotide sequences of Rf1 from the three genotypes: One had a novel allele and two had known alleles, of which one was reported to have been selected previously as a non-restoring allele at a single U.S. breeding station but not at other stations in the U.S., or in Europe or Japan, suggesting environmental sensitivity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)