Kazuyoshi Kitazaki

Unusual and Typical Features of a Novel Restorer-of-Fertility Gene of Sugar Beet (Beta vulgaris L.)

Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.

Cost of Having the Largest Mitochondrial Genome: Evolutionary Mechanism of Plant Mitochondrial Genome

The angiosperm mitochondrial genome is the largest and least gene-dense among the eukaryotes, because its intergenic regions are expanded. There seems to be no functional constraint on the size of the intergenic regions; angiosperms maintain the large mitochondrial genome size by a currently unknown mechanism. After a brief description of the angiosperm mitochondrial genome, this review focuses on our current knowledge of the mechanisms that control the maintenance and alteration of the genome. In both processes, the control of homologous recombination is crucial in terms of site and frequency. The copy numbers of various types of mitochondrial DNA molecules may also be controlled, especially during transmission of the mitochondrial genome from one generation to the next. An important characteristic of angiosperm mitochondria is that they contain polypeptides that are translated from open reading frames created as byproducts of genome alteration and that are generally nonfunctional. Such polypeptides have potential to evolve into functional ones responsible for mitochondrially encoded traits such as cytoplasmic male sterility or may be remnants of the former functional polypeptides.

Male Sterility-Inducing Mitochondrial Genomes: How Do They Differ?

Twenty-nine mitochondrial genomes from 19 angiosperm species have been completely sequenced and have been found to vary in genome size and gene content. Seven of these mitochondrial genomes are known to induce cytoplasmic male sterility (CMS), and thus can be utilized for hybrid seed production or the prevention of pollen dispersal. Genome rearrangement frequently is observed in male sterility (MS)-inducing mitochondria, but it also occurs as part of the normal inter- or intraspecific variation in male fertile (MF) mitochondria. Sequence analyses have revealed that the repertoire of genuine genes is indistinguishable between MS-inducing and MF mitochondria. Deleterious mutations appear to be rare in MS-inducing mitochondria, which may be consistent with the lack of systemic manifestation of CMS. On the other hand, several nucleotide substitutions remain to be investigated for their potential mild effects. Various mitochondrial open reading frames (ORFs) are associated with CMS (CMS-ORFs). There are some common but not strict features shared by CMS-ORFs such as their uniqueness to the CMS mitochondrial genome, their association with genes for ATPase subunits, and the hydrophobic nature of their putative translation products. It should be noted that some CMS-ORFs do not satisfy all of these criteria, and ORFs that satisfy these criteria are not necessarily associated with CMS. Therefore, it is difficult to infer the capability of MS induction of mitochondrial genomes solely from their nucleotide sequences. Morphological, physiological, and molecular biological studies suggest that multiple mechanisms cause CMS. Nuclear genes that suppress CMS have been identified. Post-transcriptional suppression of CMS-ORFs mediated by a certain class of RNA binding proteins (pentatrico peptide repeat proteins) is the predominant mechanism of fertility restoration. On the other hand, CMS suppression that is not associated with post-transcriptional suppression of CMS-ORFs has also been reported, suggesting that various types of gene products are involved in fertility restoration.

Post‐translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet (Beta vulgaris)

Genetic conflict between cytoplasmically inherited elements and nuclear genes arising from their different transmission patterns can be seen in cytoplasmic male sterility (CMS), the mitochondrion-encoded inability to shed functional pollen. CMS is associated with a mitochondrial open reading frame (ORF) that is absent from non-sterility inducing mitochondria (S-orf). Nuclear genes that suppress CMS are called restorer-of-fertility (Rf) genes. Post-transcriptional and translational repression of S-orf mediates the molecular action of Rf that encodes a class of RNA-binding proteins with pentatricopeptide repeat (PPR) motifs. Besides the PPR-type of Rfs, there are also non-PPR Rfs, but the molecular interactions between non-PPR Rf and S-orf have not been described. In this study, we investigated the interaction of bvORF20, a non-PPR Rf from sugar beet (Beta vulgaris), with preSatp6, the S-orf from sugar beet. Anthers expressing bvORF20 contained a protein that interacted with preSATP6 protein. Analysis of anthers and transgenic calli expressing a FLAG-tagged bvORF20 suggested the binding of preSATP6 to bvORF20. To see the effect of bvORF20 on preSATP6, which exists as a 250-kDa protein complex in CMS plants, signal bands of preSATP6 in bvORF20-expressing and non-expressing anthers were compared by immunoblotting combined with Blue Native polyacrylamide gel electrophoresis. The signal intensity of the 250-kDa band decreased significantly, and 200- and 150-kDa bands appeared in bvORF20-expressing anthers. Transgenic callus expressing bvORF20 also generated the 200- and 150-kDa bands. The 200-kDa complex is likely to include both preSATP6 and bvORF20. Post-translational interaction between preSATP6 and bvORF20 appears to alter the higher order structure of preSATP6 that may lead to fertility restoration in sugar beet.

The distribution of normal and male-sterile cytoplasms in Chinese sugar-beet germplasm

Forty-two Chinese sugar-beet breeding lines were evaluated for the presence of normal and male-sterile (Owen) cytoplasms using polymorphisms in the chloroplast petG-psbE region as well as in the mitochondrial minisatellite loci. The polymorphisms detected allowed the distinction of three cytoplasm types over the whole sample, one being associated with Owen cytoplasm, a second with the maintainer inbred ‘TK-81mm-O’-type cytoplasm (termed normal-1 cytoplasm) and a third with another maintainer inbred ‘NK-310mm-O’-type cytoplasm (normal-2 cytoplasm). Western blot analysis was carried out to confirm that expression of the male-sterility-associated protein (preSATP6) occurred in plants with Owen cytoplasm but not in plants with either normal-1 or normal-2 cytoplasm. Of the 42 breeding lines examined, 14 had exclusively normal (normal-1 and/or normal-2) cytoplasm and 11 had only Owen cytoplasm. The remaining 17 lines possessed both normal and Owen cytoplasms, and noticeably, some of these 17 lines have been expected to become the source of superior maintainer lines. The results thus show that molecular identification of the cytoplasm is required to avoid wasting resources on account of attempting to develop the maintainer genotype from plants with Owen cytoplasm.

Mitochondrial genome diversity in Beta vulgaris L. ssp. vulgaris (Leaf and Garden Beet Groups) and its implications concerning the dissemination of the crop

Four mitochondrial minisatellites were used to study cytoplasmic diversity in leaf and garden beet germplasm resources. Eleven multi-locus haplotypes were identified, of which one (named mitochondrial minisatellite haplotype 4, hereafter min04) was associated with male-sterile Owen cytoplasm and two others (min09 and min18), with a normal fertile cytoplasm. European leaf beet germplasm exhibited the greatest haplotype diversity, with min09 and min18 predominating. In North African leaf beet accessions, only these two haplotypes were observed, making it likely that North African accessions were descended from European genotypes. The prevalence of min18 was also noted in leaf beet from the Middle East and western Asia. Such a pattern contrasts with that found in east Asian leaf beet where the two haplotypes were extremely rare. The geographical structure of the mitochondrial haplotypes allowed us to infer possible dissemination pathways of leaf beet. Additionally, we showed that mitochondrial genome diversity was low in garden beet germplasm, with min18 being highly predominant. An explanation of this limited diversity may lie in the geographically restricted origin of as well as relatively short cultivation histories of garden beet.

A horizontally transferred tRNACys gene in the sugar beet mitochondrial genome: evidence that the gene is present in diverse angiosperms and its transcript is aminoacylated

Of the two tRNA(Cys) (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA(Cys2) (GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA(Cys2) (GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA(Cys2) (GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA(Cys1) (GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCA may have been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution.

Influence of Nitrogen Limitation and Long-Term Use of Rockwool on Nitrous Oxide Emissions in Hydroponic Systems

To mitigate Nitrous Oxide (N2O) emissions derived from Nitrogen (N) fertilizer of agroecosystems, establishment of best management protocols for cultivation is necessary. Hydroponic systems using rockwool have the potential to reduce N2O emissions; however, the effects of nutrient condition and retained N compounds in rockwool on N2O emissions remain unclear. The primary objective of our study was to understand the crucial factors behind emissions of N2O. Tomato cultivation with low levels of nutrient showed reduced growth and yield, but increased N2O emission. In contrast, growth and N2O emissions were increased by cultivation with normal levels of nutrient and used (1-yold) rockwool containing excess N compounds from the previous years cultivation. Though the long-term use of rockwool significantly enhanced seasonal N2O emission, the availability of N2O precursors NO3 − and NH4 + did not clearly explain the variation in N2O fluxes during cultivation. Rather, environmental factors, such as relative water content of rockwool in the rhizosphere, were significantly correlated to N2O emissions during cultivation under various conditions. We conclude that environmental factors most strongly influence the fate of available environmental substrates remaining in rockwool, and thereby control N2O emissions.

Metabolic Reprogramming in Leaf Lettuce Grown Under Different Light Quality and Intensity Conditions Using Narrow-Band LEDs

Light-emitting diodes (LEDs) are an artificial light source used in closed-type plant factories and provide a promising solution for a year-round supply of green leafy vegetables, such as lettuce (Lactuca sativa L.). Obtaining high-quality seedlings using controlled irradiation from LEDs is critical, as the seedling health affects the growth and yield of leaf lettuce after transplantation. Because key molecular pathways underlying plant responses to a specific light quality and intensity remain poorly characterised, we used a multi-omics–based approach to evaluate the metabolic and transcriptional reprogramming of leaf lettuce seedlings grown under narrow-band LED lighting. Four types of monochromatic LEDs (one blue, two green and one red) and white fluorescent light (control) were used at low and high intensities (100 and 300 μmol·m−2·s−1, respectively). Multi-platform mass spectrometry-based metabolomics and RNA-Seq were used to determine changes in the metabolome and transcriptome of lettuce plants in response to different light qualities and intensities. Metabolic pathway analysis revealed distinct regulatory mechanisms involved in flavonoid and phenylpropanoid biosynthetic pathways under blue and green wavelengths. Taken together, these data suggest that the energy transmitted by green light is effective in creating a balance between biomass production and the production of secondary metabolites involved in plant defence.

Molecular basis of cytoplasmic male sterility in beets: an overview

Sugarbeet cultivars are almost exclusively hybrids, which are produced using the sole source of cytoplasmic male sterility (CMS), the so-called Owen CMS. Several alternative sources of CMS have been described. One of these, I-12CMS(3), was derived from wild beets collected in Pakistan, and another CMS source, GCMS, has a cytoplasmic origin in wild sea beets from France. During the past decade, male sterility-associated mitochondrial genes have been identified in these three CMS systems. Moreover, the recent development of a variety of DNA markers has permitted the genetic mapping of nuclear restorer-of-fertility genes for both Owen and GCMS. This review focuses on the mechanism of CMS in beets.