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How do separate sexes originate and evolve? Plants provide many opportunities to address this question as they have diverse mating systems and separate sexes (dioecy) that evolved many times independently. The classic "two-factor" model for evolution of separate sexes proposes that males and females can evolve from hermaphrodites via the spread of male and female sterility mutations that turn hermaphrodites into females and males, respectively. This widely accepted model was inspired by early genetic work in dioecious white campion (Silene latifolia) that revealed the presence of two sex-determining factors on the Y-chromosome, though the actual genes remained unknown. Here, we report identification and functional analysis of the putative sex-determining gene in S. latifolia, corresponding to the gynoecium suppression factor (GSF). We demonstrate that GSF likely corresponds to a Y-linked CLV3-like gene that is specifically expressed in early male flower buds and encodes the protein that suppresses gynoecium development in S. latifolia. Interestingly, GSFY has a dysfunctional X-linked homolog (GSFX) and their synonymous divergence (dS = 17.9%) is consistent with the age of sex chromosomes in this species. We propose that female development in S. latifolia is controlled via the WUSCHEL-CLAVATA feedback loop, with the X-linked WUSCHEL-like and Y-linked CLV3-like genes, respectively. Evolution of dioecy in the S. latifolia ancestor likely involved inclusion of ancestral GSFY into the nonrecombining region on the nascent Y-chromosome and GSFX loss of function, which resulted in disbalance of the WUSCHEL-CLAVATA feedback loop between the sexes and ensured gynoecium suppression in males., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

We performed micro-X-ray fluorescence imaging of frozen-hydrated sections of a root of Pteris vittata for the first time, to the best of our knowledge, to reveal the mechanism of arsenic (As) uptake. The As distribution was successfully visualized in cross sections of different parts of the root, which showed that (i) the major pathway of As uptake changes from symplastic to apoplastic transport in the direction of root growth, and (ii) As and K have different mobilities around the stele before xylem loading, despite their similar distributions outside the stele in the cross sections. These data can reasonably explain As reduction, axially observed around the root tip in the direction of root growth and radially observed in the endodermis in the cross sections, as a consequence of the incorporation of As into the cells or symplast of the root. In addition, previous observations of As species in the midrib can be reconciled by ascribing a reduction capacity to the root cells, which implies that As reduction mechanisms at the cellular level may be an important control on the peculiar root-to-shoot transport of As in P. vittata., (© The Author(s) 2021. Published by Oxford University Press.)

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The striking sub-cellular distribution of cadmium (Cd) and zinc (Zn) in the Cd and Zn hyperaccumulator Arabidopsis halleri ssp. gemmifera was revealed by microbeam X-ray microfluorescence analysis (μ-XRF) using high-energy synchrotron radiation. Plants were grown in hydroponics with various Cd and Zn concentrations. The concentration of Cd in the aerial portions of the plants increased with increasing Zn exposure and the transportation efficiency of Cd from the root to the shoot was affected by both the Cd and Zn concentrations in the nutrient solution. The μ-XRF imaging clearly showed that Cd and Zn were preferentially accumulated in trichomes on the leaf, while the distribution of Cd in the leaf was changed by Zn treatment. It was observed that Cd treated with a higher Zn concentration (20 μM Cd + 100 μM Zn) was distributed in the mesophyll tissue at high concentrations. In addition, μ-XRF imaging clarified that the distribution of Zn inside the leaf was different from that of Cd at a cellular level. Zn was primarily distributed in the mesophyll tissue of the leaf blade. In contrast, Cd was localized in the vascular bundle of the main vein. That is, Zn was transported to mesophyll tissue from the vascular bundle more efficiently than Cd. As seen above, we were able to study the difference of the distribution of Cd and Zn, which are congeners and behave similarly, inside the plant body at the cellular level in detail by high-energy μ-XRF.

Chlorophyll is an essential molecule for acquiring light energy during photosynthesis. Mutations that result in chlorophyll retention during leaf senescence are called 'stay-green' mutants. One of the several types of stay-green mutants, Type E, accumulates high levels of chlorophyll in the pre-senescent leaves, resulting in delayed yellowing. We isolated delayed yellowing1-1 (dye1-1), a rice mutant whose yellowing is delayed in the field. dye1-1 accumulated more chlorophyll than the wild-type in the pre-senescent and senescent leaves, but did not retain leaf functionality in the 'senescent green leaves', suggesting that dye1-1 is a Type E stay-green mutant. Positional cloning revealed that DYE1 encodes Lhca4, a subunit of the light-harvesting complex I (LHCI). In dye1-1, amino acid substitution occurs at the location of a highly conserved amino acid residue involved in pigment binding; indeed, a severely impaired structure of the PSI-LHCI super-complex in dye1-1 was observed in a blue native PAGE analysis. Nevertheless, the biomass and carbon assimilation rate of dye1-1 were comparable to those in the wild-type. Interestingly, Lhcb1, a trimeric LHCII protein, was highly accumulated in dye1-1, in the chlorophyll-protein complexes. The high accumulation of LHCII in the LHCI mutant dye1 suggests a novel functional interaction between LHCI and LHCII.

We isolated a cold sensitive virescent1 (csv1) mutant from a rice (Oryza sativa L.) population mutagenized by carbon ion irradiation. The mutant exhibited chlorotic leaves during the early growth stages, and produced normal green leaves as it grew. The growth of csv1 plants displayed sensitivity to low temperatures. In addition, the mutant plants that were transferred to low temperatures at the fifth leaf stage produced chlorotic leaves subsequently. Genetic and molecular analyses revealed translocation of a 13-kb genomic fragment that disrupted the causative gene (CSV1; LOC_Os05g34040). CSV1 encodes a plastid-targeted oxidoreductase-like protein conserved among land plants, green algae, and cyanobacteria. Furthermore, CSV1 transcripts were more abundant in immature than in mature leaves, and they did not markedly increase or decrease with temperature. Taken together, our results indicate that CSV1 supports chloroplast development under cold stress conditions, in both the early growth and tillering stages in rice.

Background: In addition to classically activated macrophages that have effector roles in tissue injury, alternatively activated M2 macrophages are involved in the resolution of inflammation in animal models of kidney disease. To clarify the clinical relevance of macrophage phenotypes in human glomerular diseases, we evaluated the renal accumulation of macrophages and plasma and urine levels of CD163, an M2 marker, in lupus nephritis (LN) patients., Methods: Kidney biopsies and plasma and urine samples were obtained from LN patients who underwent renal biopsy between 2008 and 2012. CD163 + , CD68 + and CD204 + cells were counted in paraffin-embedded and frozen sections. LN histological activity was evaluated semiquantitatively using the biopsy activity index. Plasma and urinary soluble CD163 (sCD163) concentrations were also measured and evaluated for their significance as potential LN biomarkers., Results: Immunohistological analysis of glomeruli from LN patients revealed that >60% of CD68 + macrophages had merged with CD163 + cells. The increased number of glomerular CD163 + macrophages was correlated with LN severity, as determined by the biopsy active index (r = 0.635). Urinary (u-) sCD163 level was strongly correlated with glomerular CD163 + cell counts and histological disease score as well as urinary monocyte chemoattractant protein 1 levels (r = 0.638 and 0.592, respectively). Furthermore, the u-sCD163 level was higher in patients with active LN than in those with other diseases., Conclusions: Glomerular CD163 + macrophages are the predominant phenotype in the kidneys of lupus patients. These findings indicate that the u-sCD163 level can serve as a biomarker for macrophage-dependent glomerular inflammation in human LN., (© The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Euglena gracilis is a common phytoplankton species, which also has motile flagellate characteristics. Recent research and development has enabled the industrial use of E. gracilis and selective breeding of this species is expected to further expand its application. However, the production of E. gracilis nuclear mutants is difficult because of the robustness of its genome. To establish an efficient mutation induction procedure for E. gracilis, we employed Fe-ion beam irradiation in the RIKEN RI beam factory. A decrease in the survival rate was observed with the increase in irradiation dose, and the upper limit used for E. gracilis selective breeding was around 50 Gy. For a practical trial of Fe-ion irradiation, we conducted a screening to isolate high-temperature-tolerant mutants. The screening yielded mutants that proliferated faster than the wild-type strain at 32 °C. Our results demonstrate the effectiveness of heavy-ion irradiation on E. gracilis selective breeding.

Plant shoot organs such as stems, leaves and flowers are derived from specialized groups of stem cells organized at the shoot apical meristem (SAM). Organogenesis involves two major processes, namely cell proliferation and differentiation, whereby the former contributes to increasing the cell number and the latter involves substantial increases in cell volume through cell expansion. Co-ordination between the above processes in time and space is essential for proper organogenesis. To identify regulatory factors involved in proper organogenesis, heavy-ion beam-irradiated de-etiolated (det) 3-1 seeds have been used to identify striking phenotypes in the A#26-2; det3-1 mutant. In addition to the stunted plant stature mimicking det3-1, the A#26-2; det3-1 mutant exhibited stem thickening, increased floral organ number and a fruit shape reminiscent of clavata (clv) mutants. DNA sequencing analysis demonstrated that A#26-2; det3-1 harbors a mutation in the CLV3 gene. Importantly, A#26-2; det3-1 displayed cracks that randomly occurred on the main stem with a frequency of approximately 50%. Furthermore, the double mutants clv3-8 det3-1, clv1-4 det3-1 and clv2-1 det3-1 consistently showed stem cracks with frequencies of approximately 97, 38 and 35%, respectively. Cross-sections of stems further revealed an increase in vascular bundle number, cell number and size in the pith of clv3-8 det3-1 compared with det3-1. These findings suggest that the stem inner volume increase due to clv mutations exerts an outward mechanical stress; that in a det3-1 background (defective in cell expansion) resulted in cracking of the outermost layer of epidermal cells., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Silene latifolia is a dioecious plant with heteromorphic XY sex chromosomes. Previous studies of sex chromosome-linked genes have suggested a gradual divergence between the X-linked and the Y-linked genes in proportion to the distance from the pseudoautosomal region. However, such a comparison has yet to be made for the noncoding regions. To better characterize the nonrecombining region of the X and Y chromosomes, we sequenced bacterial artificial chromosome clones containing the sex chromosome-linked paralogs SlAP3X and SlAP3Y, including 115 kb and 73 kb of sequences, respectively, flanking these genes. The synonymous nucleotide divergence between SlAP3X and SlAP3Y indicated that recombination stopped approximately 3.4 million years ago. Sequence homology analysis revealed the presence of six long terminal repeat retrotransposon-like elements. Using the nucleotide divergence calculated between left and right long terminal repeat sequences, insertion dates were estimated to be 0.083-1.6 million years ago, implying that all elements detected were inserted after recombination stopped. A reciprocal sequence homology search facilitated the identification of four homologous noncoding DNA regions between the X and Y chromosomes, spanning 6.7% and 10.6% of the X chromosome-derived and Y chromosome-derived sequences, respectively, investigated. Genomic Southern blotting and fluorescence in situ hybridization showed that the noncoding DNA flanking SlAP3X/Y has homology to many regions throughout the genome, regardless of whether they were homologous between the X and Y chromosomes. This finding suggests that most noncoding DNA regions rapidly lose their counterparts because of the introduction of transposable elements and indels (insertion-deletions) after recombination has stopped.

The dioecious plant Silene latifolia has heteromorphic sex chromosomes, and comparison of the positions of sex-linked genes indicates that at least three large inversions have occurred during the evolution of the Y chromosome. In this article, we describe the isolation of a new sex-linked gene from S. latifolia, which provides new information on the evolution of this plant's young sex chromosomes. By using reverse-transcription polymerase chain reaction degenerate primers based on the Arabidopsis thaliana sequence of WUSCHEL, a flower-development gene, we found two copies in S. latifolia, which we named SlWUS1 and SlWUS2. Southern blot and genetic segregation analysis showed that SlWUS1 is located on the X chromosome and SlWUS2 is autosomal. No Y-linked copy of SlWUS1 was found by either Southern blot analysis under low-stringency conditions or polymerase chain reaction with degenerate primers, so we conclude that SlWUS1 probably has no Y-linked homolog. It is unknown whether the Y chromosome lost the SlWUS1 copy by degeneration of this individual gene or whether deletion of a larger genome region was involved. Several tests lead us to conclude that dosage compensation has not evolved for this sex-linked gene. We mapped the ortholog in the nondioecious relative S. vulgaris (SvWUS1), to compare the location in a species that has no history of having sex chromosomes. SvWUS1 maps to the same linkage group as other fully X-linked genes, indicating that it was not added to the X, but was lost from the Y. Its location differs in the maps from the two species, raising the possibility that the X chromosome, as well as the Y, may have been rearranged.

Silene latifolia is a well-studied model system for plant XY sex determination. Three maleness factors are thought to function on the Y chromosome, gynoecium suppression factor (GSF), stamen-promoting factor (SPF), and male fertility factor (MFF), and their deletions result in hermaphrodites, anther defects, and pollen defects, respectively. Although a framework map of the Y chromosome exists, the sex determination genes have not been identified, and no markers close enough to potentially be used for BAC library screening are yet available. The analysis of Y deletion mutants by Y-chromosome-specific STS markers is an efficient way to isolate sex determination regions, but more Y-specific STS markers are needed to accelerate the exploration of sex determination factors. Herein, we report a marker design method that uses simple sequence repeats, which is especially effective on the Y chromosome of S. latifolia because it contains many simple sequence repeats. Six new Y-chromosome-specific STS markers were obtained, SmicSy1-6. These were used to detect relatively small Y deletion sites in heavy-ion beam irradiation-induced mutants. The mapping of male sex determination regions was narrowed down by using more markers and smaller-sized Y deletion mutants. One new marker, SmicSy6, is a proximal marker to SPF and, thus, a second index for SPF. The region including SPF is thought to be located between two SPF proximal markers. The flower phenotype correlates with the deletion size of SPF using SPF proximal markers. These findings represent new progress in isolating the sex determination factor, which has been studied for more than 50 years.

In vivo X-ray analysis utilizing synchrotron radiation was performed to investigate the distribution and oxidation state of arsenic in the gametophytes of two hyperaccumulators, Pteris vittata L. and Pteris cretica L., and an arsenic-accumulating fern, Athyrium yokoscense in the several growth stages from germination. The distribution of arsenic in P. vittata changed through the development of the plant tissues as follows. In two-week-old gametophyte, arsenic was mainly present along the rhizoid. In the one-month-old gametophyte with reproductive organs, arsenic was accumulating uniformly in the sheet of cells, except in the reproductive area. After fertilization, arsenic was observed in the aboveground part of the sporophyte structures. P. cretica and A. yokoscense showed different distributions, respectively. P. cretica showed an accumulation of arsenic in the reproductive area, in contrast to P. vittata, before fertilization, while arsenic was observed in the aboveground part of the sporophyte after fertilization. A. yokoscense showed an accumulation of arsenic along the rhizoids before fertilization, while it was present mainly along the roots of the sporophyte after fertilization. Reduced arsenic (As(iii)) was observed in all stages and in all tissues of P. vittata gametophytes. Further, a reduction of arsenic was commonly observed among the three ferns, although arsenic was bounded to sulfur in A. yokoscense. These findings may be related to their own reproductive process or to detoxification mechanism. They provide basic information for the understanding of arsenic hyperaccumulation in these ferns, leading to further application of these gametophyte systems.

Carpel suppression is a trigger for sexual dimorphism in the dioecious plant Silene latifolia. To clarify what kind of genes are involved in carpel suppression in this species, we generated a bisexual mutant, R025, by C-ion beam irradiation. R025 produces bisexual flowers with a mature gynoecium and mature stamens. Genetic analysis of R025 attributed the bisexual trait to mutations on the Y chromosome. Scanning electron microscopy (SEM) analysis of early floral development revealed that the carpel size of R025 was different from that of wild-type males in spite of the male background in R025. We also identified an S. latifolia CLAVATA1-like gene (SlCLV1) as a candidate of the CLAVATA-WUSCHEL (CLV-WUS) pathway. Two separate pathways, the CLV-WUS pathway and the CUP-SHAPED COTYLEDON (CUC)-SHOOT MERISTEMLESS (STM) pathway, contribute to carpel development in the Arabidopsis floral meristem. SlSTM1 and SlSTM2 (orthologs of STM) and SlCUC (an ortholog of CUC1 and CUC2) have already been identified in S. latifolia. We therefore examined the expression patterns of SlCLV1, SlSTM (SlSTM1 and SlSTM2) and SlCUC in young flowers of R025 and wild-type males and females. The expression patterns of the three genes in the two pathways differ between the wild-type male and the bisexual mutant, and the differences in expression patterns of the three genes occur at the same stage. These results suggest that in addition to SlSTM1, SlSTM2 and SlCUC, SlCLV1 is also involved in carpel suppression in S. latifolia. They also suggest that a gynoecium-suppressing factor (GSF), which is lost in the R025 Y chromosome, acts on an upstream gene that is common to the two pathways, triggering sexual dimorphism in S. latifolia.

Symmetric chloroplast division requires a prokaryote-derived division regulator protein MinD, whose subchloroplastic localization remains to be completely established. We investigated the localization and functionality of AtMinD1 (Arabidopsis thaliana MinD) fused with a dual hemagglutinin epitope (dHA) or a yellow fluorescent protein (YFP). AtMinD1-dHA, which successfully complemented the arc11/atminD1 mutant phenotype, was predominantly located at the envelope membrane and the mid-chloroplast constriction site. Meanwhile, AtMinD1-YFP was non-functional and showed suborganellar localization partly similar to that of AtMinD1-dHA. This prompts us to reevaluate earlier transgenic and transient expression studies using fluorescent protein-tagged AtMinD1.

Chloroplast division involves the tubulin-related GTPase FtsZ that assembles into a ring structure (Z-ring) at the mid-chloroplast division site, which is where invagination and constriction of the envelope membranes occur. Z-ring assembly is usually confined to the mid-chloroplast site by a well balanced counteraction of the stromal proteins MinD and MinE. The in vivo mechanisms by which FtsZ nucleates at specific sites, polymerises into a protofilament and organizes a closed ring of filament bundles remain largely unknown. To clarify the dynamic aspects of FtsZ, we developed a living cell system for simultaneous visualisation of various FtsZ configurations, utilising the Arabidopsis thaliana overexpressor and mutant of the MinE (AtMinE1) gene, which were modified to weakly express green fluorescent protein (GFP) fused to AtFtsZ1-1. Time-lapse observation in the chloroplasts of both plants revealed disorderly movement of the dots and short filaments of FtsZ. The short filaments often appeared to emanate from the dots and to converge with a long filament, producing a thick cable. In the AtMinE1 overexpressor, we also observed spirals along the longitudinal axis of the organelle that often rolled the closed rings together. In the atminE1 mutant, we visualised the 'isolated' rings with a maximum diameter of approximately 2 mum that did not encircle the organelle periphery, but appeared to be suspended in the stroma. Our observations further demonstrated heterogeneity in chloroplast shapes and concurrently altered configurations of FtsZ in the mutant.

To elucidate the mechanism(s) underlying dioecious flower development, the present study analyzed a SUPERMAN (SUP) homolog, SlSUP, which was identified in Silene latifolia. The sex of this plant is determined by heteromorphic X and Y sex chromosomes. It was revealed that SlSUP is a single-copy autosomal gene expressed exclusively in female flowers. Introduction of a genomic copy of SlSUP into the Arabidopsis thaliana sup (sup-2) mutant complemented the excess-stamen and infertile phenotypes of sup-2, and the overexpression of SlSUP in transgenic Arabidopsis plants resulted in reduced stamen numbers as well as the suppression of petal elongation. During the development of the female flower in S. latifolia, the expression of SlSUP is first detectable in whorls 2 and 3 when the normal expression pattern of the B-class flowering genes was already established and persisted in the stamen primordia until the ovule had matured completely. In addition, significant expression of SlSUP was detected in the ovules, suggestive of the involvement of this gene in ovule development. Furthermore, it was revealed that the de-suppression of stamen development by infection of the S. latifolia female flower with Microbotryum violaceum was accompanied by a significant reduction in SlSUP transcript levels in the induced organs. Taken together, these results demonstrate that SlSUP is a female flower-specific gene and suggest that SlSUP has a positive role in the female flower developmental pathways of S. latifolia.

Chloroplast division comprises a sequence of events that facilitate symmetric binary fission and that involve prokaryotic-like stromal division factors such as tubulin-like GTPase FtsZ and the division site regulator MinD. In Arabidopsis, a nuclear-encoded prokaryotic MinE homolog, AtMinE1, has been characterized in terms of its effects on a dividing or terminal chloroplast state in a limited series of leaf tissues. However, the relationship between AtMinE1 expression and chloroplast phenotype remains to be fully elucidated. Here, we demonstrate that a T-DNA insertion mutation in AtMinE1 results in a severe inhibition of chloroplast division, producing motile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor) plants, dividing chloroplasts possess either single or multiple FtsZ rings located at random intervals and showing constriction depth, mainly along the chloroplast polarity axis. The AtMinE1 sense plants displayed equivalent chloroplast phenotypes to arc11, a loss-of-function mutant of AtMinD1 which forms replicating mini-chloroplasts. Furthermore, a certain population of FtsZ rings formed within developing chloroplasts failed to initiate or progress the membrane constriction of chloroplasts and consequentially to complete chloroplast fission in both AtMinE1 sense and arc11/atminD1 plants. Our present data thus demonstrate that the chloroplast division site placement involves a balance between the opposing activities of AtMinE1 and AtMinD1, which acts to prevent FtsZ ring formation anywhere outside of the mid-chloroplast. In addition, the imbalance caused by an AtMinE1 dominance causes multiple, non-synchronous division events at the single chloroplast level, as well as division arrest, which becomes apparent as the chloroplasts mature, in spite of the presence of FtsZ rings.

We have developed an efficient system to detect and analyze DNA mutations induced by heavy-ion beams in Arabiopsis thaliana. In this system, a stable transgenic Arabidopsis line that constitutively expresses a yellow fluorescent protein (YFP) by a single-copy gene at a genomic locus was constructed and irradiated with heavy-ion beams. The YFP gene is a target of mutagenesis, and its loss of function or expression can easily be detected by the disappearance of YFP signals in planta under microscopy. With this system, a (12)C(6+)-induced mutant with single deletion and multiple base changes was isolated.

Mammalian promoters are categorized into TATA and CpG-related groups, and they have complementary roles associated with differentiated transcriptional characteristics. While the TATA box is also found in plant promoters, it is not known if CpG-type promoters exist in plants. Plant promoters contain Y Patches (pyrimidine patches) in the core promoter region, and the ubiquity of these beyond higher plants is not understood as well. Sets of promoter sequences were utilized for the analysis of local distribution of short sequences (LDSS), and approximately one thousand octamer sequences have been identified as promoter constituents from Arabidopsis, rice, human and mouse, respectively. Based on their localization profiles, the identified octamer sequences were classified into several major groups, REG (Regulatory Element Group), TATA box, Inr (Initiator), Kozak, CpG and Y Patch. Comparison of the four species has revealed three categories: (i) shared groups found in both plants and mammals (TATA box), (ii) common groups found in both kingdoms but the utilized sequence is differentiated (REG, Inr and Kozak) and (iii) specific groups found in either plants or mammals (CpG and Y Patch). Our comparative LDSS analysis has identified conservation and differentiation of promoter architectures between higher plants and mammals.

Sialic acids are widely distributed among living creatures, from bacteria to mammals, but it has been commonly accepted that they do not exist in plants. However, with the progress of genome analyses, putative gene homologs of animal sialyltransferases have been detected in the genome of some plants. In this study, we cloned three genes from Oryza sativa (Japanese rice) that encode sialyltransferase-like proteins, designated OsSTLP1, 2, and 3, and analyzed the enzymatic activity of the proteins. OsSTLP1, 2, and 3 consist of 393, 396, and 384 amino acids, respectively, and each contains sequences similar to the sialyl motifs that are highly conserved among animal sialyltransferases. The recombinant soluble forms of OsSTLPs produced by COS-7 cells were analyzed for sialyltransferase-like activity. OsSTLP1 exhibited such activity toward the oligosaccharide Galbeta1,4GlcNAc and such glycoproteins as asialofetuin, alpha1-acid glycoprotein, and asialo-alpha1-acid glycoprotein; OsSTLP3 exhibited similar activity toward asialofetuin; and OsSTLP2 exhibited no sialyltransferase-like activity. The sialic acid transferred by OsSTLP1 or 3 was linked to galactose of Galbeta1,4GlcNAc through alpha2,6-linkage. This is the first report of plant proteins having sialyltransferase-like activity.

Gametophytes of the fern Adiantum capillus-veneris L. were mutagenized by heavy ion beam irradiation and screened for mutants lacking chloroplast avoidance movement under high intensity blue light. Mutants recovered include several with small deletions in the AcPHOT2 gene. The avoidance movement response in these mutants could be restored by transient expression of non-mutant AcPHOT2 cDNA, indicating that the chloroplast avoidance movement in this fern is mediated by the Acphot2 protein. Further functional analyses of the Acphot2 protein were performed using this transient assay for chloroplast avoidance movement. The results obtained suggest that the LOV2, but not the LOV1, domain of Acphot2 is essential for avoidance movement, and that several residues in the C-terminus of the kinase domain contribute to the avoidance response. The rate of dark reversion of the photo-activated LOV2 domain, which was calculated photometrically, was too fast to account for the lifetime of phot2 signal estimated from physiological responses. However, the rate of dark reversion of the combined domains of LOV1 and LOV2 did correspond to the lifetime of the signal, suggesting that LOV1 might have some function in this response, although it is not essential for playing a role as a photoreceptor.

Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.

The effects of heavy ion particle irradiation on meiosis and reproductive development in the nematode Caenorhabditis elegans were studied. Meiotic pachytene nuclei are significantly resistant to particle irradiation by the heavy ions carbon and argon, as well as to X-rays, but not UV, whereas diplotene to diakinesis stage oocytes and early embryonic cells are not. Chromosomal abnormalities appear in mitotic cells and in maturing oocytes irradiated with heavy ion particles during the diplotene to the early diakinesis stages, but not in oocytes irradiated during the pachytene stage. The pachytene nuclei of ced-3 mutants, which are defective in apoptosis, are similarly resistant to ionizing radiation, but pachytene nuclei depleted for Ce-atl-1 (ataxia-telangiectasia like 1) or Ce-rdh-1/rad-51 are more sensitive. Pachytene nuclei thus appear to effectively repair heavy ion-induced DNA damage by the meiotic homologous recombination system.

Understanding the role of 'epigenetic' changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development.

Heavy-ion irradiation is a new method of mutation breeding to produce new cultivars. We established the application of this method in rice plants to obtain mutants. Rice seeds were irradiated by C or Ne ions (135MeV/u) with a LET (linear energy transfer) of 22.7 or 64.2 keV/microm, respectively. Chlorophyll-deficient mutants (CDM) segregated in M2 progeny were albino, pale-green, yellow or striped-leave phenotypes. The highest rate of CDM with C-ion irradiation, 7.31%, was obtained at 40 Gy among the doses examined. Ne-ion irradiation gave the highest rate, 11.6%, at 20 Gy. We used the RLGS (Restriction Landmark Genomic Scanning) method to analyze DNA deletion in an albino mutant genome. Not I-landmark RLGS profiles detected about 2000 spots in rice. We found that one of the polymorphic spots was strongly linked to the albino phenotypic mutant derived from deleting of a DNA fragment, and demonstrated the high ability to detect of polymorphic regions by the RLGS method.