The plasma membrane H⁺-ATPase in plant cells functions as a critical transporter, propelling protons against an electrochemical gradient. This action generates pH and potential differences across the plasma membrane, contributing to various plant physiological processes, including cell growth, nutrient absorption, and stomatal movements. Plant plasma membrane H⁺-ATPase activity is tightly regulated in response to a multitude of abiotic and biotic environmental cues. Recent studies have shed light on the multi-level regulation of H⁺-ATPase, extending beyond its proton transport activity and recognizing the importance of its abundance at the plasma membrane, achieved by a balance of endocytosis and exocytosis in response to environmental factors. This review provides an overview of the cell biological regulation of plasma membrane H⁺-ATPase by balancing its exocytosis and endocytosis. As key factors in regulation of the abundance of the plasma membrane H⁺-ATPase at the plasma membrane, the membrane trafficking proteins PATROL1 and SYP132 have been identified. The intracellular dynamics and functions are suggested to be tightly associated with the environmental response of plant cells. Despite substantial progress in deciphering the intracellular traffic regulation of the plasma membrane H⁺-ATPase, a comprehensive molecular understanding remains elusive. Elucidating the molecular pathways linking environmental factors with the regulation of membrane trafficking is pivotal for comprehending the cell biological regulation of environmental responses in plants.

Prof. Tsunewaki dedicated his entire scientific career toward understanding the genetic effects and the diversity of “plasmon”, the whole cytoplasmic genetic system, in the common wheat Triticum and its relative Aegilops. He concluded his life’s work with a set of final experiments to test the genetic autonomy of the plasmon in this complex. They were represented by two interconnected studies. First, the examination of the persistence of genetic effects of Ae. caudata plasmon on the phenotype of the common wheat, Triticum aestivum strain “Tve” (genome: AABBDD) during 63 generations of repeated backcrosses with Tve pollen. The second study involved the reconstruction of an Ae. caudata strain by replacing the nuclear genome of the alloplasmic Tve mentioned above, (caudata)-Tve SB₅₀, with the genome (CC) of Ae. caudata. In this experiment, he tested whether there are any differences between the native plasmon of Ae. caudata and the caudata plasmon that had remained in common wheat for more than 60 generations. This paper reviews the outline and background of the last work of Prof. Koichiro Tsunewaki.

Genetic information of plants is preserved in the tripartite organellar, i.e., nucleus, plastome, and chondriome. Each organelle contains its DNA. To characterize the structures of plastome and chondriome in wheat species, the entire sequence of chloroplast DNA and mitochondrial DNA of Chinese Spring wheat was determined. Chloroplast DNA of Chinese Spring wheat conferred the 134,545 bp circular molecule with 20,703 bp inverted repeats and the same size and gene content as those of the rice and maize, indicating the conservation of the plastome among angiosperms. This conservation of the plastome among plant species allows us to investigate the variation of the plastome and their phylogenetic relationships among wheat species in the maternal lineage. On the other hand, the mitochondrial genome of angiosperms revealed complex structures. Consequently, cosmid walking of the mitochondrial DNA in the Chinese Spring wheat was carried out to construct the master copy that contains all reported genes in it. Complex features of the wheat mitochondrial genome through the recombination were able to be clarified. Paternal transmissions of mitochondrial DNAs were disclosed. To conduct the plasmon investigation of wheat species, alloplasmic lines of common wheat, which were systematically produced by Prof. Tsunewaki were indispensable. We sincerely acknowledge his great contribution to the plasmon study of wheat species.

In the tribe Triticeae, systematic studies using alloplasmic lines have greatly facilitated our understanding of cytoplasmic genome inheritance, its structural and functional diversity, and interaction with nuclear genomes. Effective utilization of these valuable experimental tools produced by combining given nuclear genomes with cytoplasmic genomes from various related species has provided ample evidence supporting the presence and roles of a genetic system controlling nucleus-cytoplasm compatibility operating particularly in the lineages of allopolyploid speciation. This review focused on the current state of our knowledge of the nucleus-cytoplasm compatibility system. It also emphasized the necessity for further research toward more precise identification and understanding of its complex genetic regulatory mechanism and possible practical application in agriculture.

Photoperiod-sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm under long-day (LD) photoperiods (≧15 h) has been proposed as a two-line system for producing hybrid varieties of common wheat (Triticum aestivum). PCMS lines are maintained by self-pollination under short-day (SD) conditions (＜15 h), while outcrossing PCMS plants to a T. aestivum pollinator line under LD conditions produces hybrid seeds. In this system, it is not necessary to have maintainer lines in order to sustain the PCMS lines. Several promising elite PCMS lines with a Japanese wheat cultivar ‘Fukuotome’ genetic background have been developed; these lines show high cross-pollination fertility and high male sterility under LD conditions, and high seed fertility under SD conditions. In addition, the European wheat cultivar ‘Fortunato’ was found to be suitable for use in the PCMS system. Through the use of the PCMS elite lines and the selected pollinator line, F₁ seeds have been produced under LD conditions in Hokkaido, Japan, and the agronomic characters of F₁ hybrids have been examined under SD conditions in Fukui, Japan. The results indicate the practicality of using this PCMS system for hybrid wheat production in Japan.

Alloplasmic lines or cytoplasmic substitution lines of bread wheat (Triticum aestivum) that have cytoplasm from a related wild Aegilops species through recurrent backcrossing exhibit several useful characteristics for wheat breeding. Using an alloplasmic line initially developed by Professor Tsunewaki, we derived new alloplasmic lines for 14 Japanese bread wheat cultivars with Aegilops mutica cytoplasm to examine the effects of the cytoplasm on agronomic characters. All alloplasmic lines showed delayed heading time (4 to 17 days in the field) compared with the euplasmic lines, and the degree of heading delay depended on VERNALIZATION1 (VRN1) genotype. In spring wheat cultivars such as ‘Minaminokaori’ that carry the dominant VRN-A1 allele, the degree of heading delay due to the alien cytoplasm was large; by contrast, in winter wheat cultivars such as ‘Haruibuki’ that carry recessive vrn1 alleles in three homoeologous genes, the heading delay was small. Compared with euplasmic lines, the alloplasmic lines generally showed increased spikelet number per spike, but decreased floret number per spikelet, leading to decreased grain number per spike (GNS). However, GNS varied depending on genotype; in the alloplasmic lines of ‘Nanbukomugi,’ ‘Nebarigoshi,’ and ‘Fukusayaka,’ no decrease in GNS occurred. Furthermore, the ‘Nebarigoshi’ and ‘Fukusayaka’ alloplasmic lines could suppress the decrease in spike number per plant during winter due to delayed flowering. These alloplasmic lines will be useful for the development of varieties adapted to global warming.

Submergence and waterlogging stresses inhibit wheat growth and cause yield loss by affecting photosynthesis, respiration, and transpiration through changing antioxidant systems and reducing accumulation and remobilization of photosynthetic products. Nucleus-cytoplasm (NC) interactions have been shown to affect sensitivity to submergence stress in wheat. We performed RNA-seq analysis of genes that show differential expression in response to submergence stress imposed during imbibition and germination of wheat seeds. W employed NC hybrids with low (T-type plasmon) and high (U-type plasmon) sensitivity and their wheat nuclear donor cv. CS. In addition, gene ontology (GO) enrichment analysis was performed on differentially expressed genes (DEGs) to estimate their transcriptional characteristics. Among DEGs down-regulated by submergence stress, ones with GO terms such as “response to oxygen-containing compound”, “response to acid chemical” and “response to jasmonic acid” were significantly enriched in all lines. On the other hand, GO terms “related to cell wall” were highly enriched in genes up-regulated in NC hybrid with U-type plasmon. Our results revealed comprehensive gene expression patterns induced under submergence stress in what. It was also suggested that the expressions of different groups of genes were differentially regulated depending on different cytoplasm types in response to submergence stress in wheat.

Meiotic study revealed the presence of B chromosomes in two species of Trigonella L. Meiotic abnormalities, pollen fertility, and chiasma frequency were taken into consideration to investigate the effects of B chromosomes. Several meiotic abnormalities were observed as a consequence of chromatin stickiness and abnormal spindle activity such as asynchronous condition, precocious movement, clumping, chromosome breakage, vagrant chromosome, cytomixis, polar tendency, unequal distribution, laggard chromosome, tri-polarity and disturbed polarity. Stickiness of the chromosome was found to be dominant over the other meiotic abnormalities in both metaphase-I/II and anaphase-I/II. Here, B chromosomes showed a neutralizing effect on meiotic abnormality and had an inert control over chiasma frequency thus producing fertile pollen. Nowadays, studying B chromosomes became an important fact because the contribution of B chromosome in plants is not well known to all.

Spirotetramat (SPT) is a new insecticide derivative of tetronic acid used extensively in agriculture to enhance the protection of major food crops against scales and aphids. This study aims to determine SPT’s cytotoxic and genotoxic effects using two model plants, Allium cepa and Vicia faba. This evaluation consists of studying the root growth, morphology, and color and the parameters of mitotic index (MI) and chromosome aberrations (CAs) as accurate toxicity markers. Our results showed a significant decrease in mean root length in A. cepa from the 0.0025% concentration. In contrast, a substantial reduction in mean root length in V. faba was recorded only at the 0.02% concentration. Furthermore, the MI was decreased proportionally with increasing concentration and time of exposure to SPT. A significant increase in CAs was observed in A. cepa and V. faba from the 0.0025% concentration after 24 h of treatment. The substantial reduction in MI and abundance of CAs indicated strong genotoxicity of SPT. From the data obtained, it can be concluded that SPT could be absorbed by the exposed plant or other non-target organisms in the proximity, causing damage to agricultural plants, affecting their genomes, and harming the environment.

Sodium valproate (VPA) is an epidrug that inhibits histone deacetylases and favors histone acetylation and DNA demethylation, thereby inducing chromatin remodeling in several cell types. Although VPA induced heterochromatin decondensation in the hemipteran Triatoma infestans, this phenomenon was not favored by histone acetylation. Considering that DNA demethylation leading to chromatin unpacking is another consequence of VPA action in several organisms, we used immunofluorescence to investigate whether cytosine methylation was the target of VPA action that affected heterochromatin remodeling in T. infestans. In addition, Fourier transform infrared (FTIR) analysis was used to evaluate the participation of cytosine in T. infestans DNA. Immunofluorescence results indicated the absence of 5-methylcytosine signals in heterochromatin, but the presence of these signals in euchromatin, where demethylation was promoted by VPA. The FTIR spectra of T. infestans global DNA exhibited marks indicative of AT richness and a low representation of cytosine contribution. In conclusion, the chromatin remodeling event previously reported in the heterochromatin compartment of T. infestans in response to VPA, which was not due to histone acetylation, was also not due to DNA cytosine demethylation.

In this study, microspore formation (microsporogenesis), gamete formation (microgametogenesis), and pollen features of Silene sangaria, a species endemic to Turkey, was examined cytoembryologically and histochemically. The species is distributed along the Black Sea coast of Turkey. The materials were collected from the coast of Igneada village (Kirklareli province). The anthers, separated by size, were passed through ethyl alcohol concentration series, and embedded in historesin. Sections were sliced using a rotary microtome and stained with toluidine blue O for general histological observations, Coomassie brilliant blue for proteins, and periodic acid-Schiff for insoluble polysaccharides. The aceto-orcein squash technique was used for cytological observations, and lactophenol-aniline blue solution was used to assess pollen viability. The anthers of S. sangaria are tetrasporangiate, and its anther wall development is of basic type. The tapetum is secretory type, and cytokinesis is simultaneous type. As a result of meiotic division of microspore mother cells, 43.5% decussate, 28.2% rhomboidal, 21.1% tetrahedral and 7.2% isobilateral tetrads occur. The released microspores first pass through the first pollen mitosis to form vegetative and generative cells, then the generative cell passes through the second pollen mitosis to form two sperm cells. Pollen grains are three-celled when released from the anther. Pollen viability rate is high (91.82%). Mature pollen grain contains a high concentration of insoluble polysaccharide and protein.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been extensively studied for potential clinical use. Recently, increasing attention is being paid to the relationship between p53 and BMSCs differentiation. In this study, we investigated the effects of p53 on the alveolar epithelial differentiation of BMSCs. Rat BMSCs (rBMSCs) were cultured with and without hyperoxia-damaged lung tissue to observe their differentiation process in vitro. Furthermore, p53 expression was inhibited by pifithrin-α during rBMSCs differentiation. Transmission electron microscopy, immunofluorescence, luciferase assay, and an analysis of cell proliferation and apoptosis levels were performed to examine the changes in rBMSCs. We found that rBMSCs in vitro exhibited alveolar epithelial differentiation when co-cultured with damaged lung tissue, and cell apoptosis, cell proliferation, and p53 levels of rBMSCs were significantly altered during the differentiation process. Furthermore, when p53 was inhibited in rBMSCs by pifithrin-α (a specific p53 inhibitor), apoptosis was alleviated, cell proliferation was promoted, and the potential alveolar epithelial differentiation of rBMSCs was blocked. Our results suggest that p53 can effectively regulate alveolar epithelial differentiation of rBMSCs. These results provide the basis for developing more effective cellular clinical therapies for lung diseases in further research.

Endoplasmic reticulum (ER) stress due to aging is one of the critical factors that inhibit stem cell differentiation and cause differentiation failure. However, some ER stress-related proteins play important roles in the regulation of stem cell differentiation and are upregulated during differentiation. Thus, ER stress is associated with both positive and negative regulation of adipocyte differentiation. Nevertheless, it remains unclear whether the upregulation of ER stress-related proteins is a cause or an effect of differentiation. This study investigated the effect of the ER stress signaling pathway on mesenchymal stem cell (MSC) differentiation. Before stimulating their differentiation, MSCs were transiently treated with tunicamycin to induce a mild ER stress, which does not induce apoptosis. We found that activating transcription factor 4 (ATF4) was upregulated by ER stress and/or adipocyte differentiation stimulation. This increase in ATF4 expression resulted in either an increase or a decrease in tribbles-related protein 3 (TRB3) expression. Our findings suggest that TRB3 downregulation is required for adipocyte differentiation. These results indicate that TRB3 might be a key protein that switches on cells in response to ER stress or adipocyte differentiation.

The popular ornamental flowering plant Dianthus hybrida cv. Telstar Scarlet has been found to exhibit two populations, each with distinct flower morphology: female-like and hermaphroditic. In this work, flower development of D. hybrida was characterized through scanning electron microscopy, light- or stereo- microscopy, from flower meristem formation to the fully matured, open flower. The difference between hermaphrodite and female-like plants was initially marked by the phenomenon of anther shrinking in the latter, which was closely associated with pollen shrinking phenomenon, and soon followed by differential elongation rates of pistils and stamens. Furthermore, the female-like anther, albeit exhibiting delay in pollen development initially, could produce microspores, resembling its hermaphrodite counterparts at some point, before shrinking. However, female-like’s pollen and cell wall size never became as large as the hermaphrodite’s.

Flowering plants have immotile sperm cells that are delivered to the female gametes by the pollen tube cell. Although the sperm chromatin in flowering plants is highly condensed like animal sperm chromatin, it is generally accepted that the transcription status in the sperm chromatin of flowering plants is active because of the existence of active gene promoters and active histone modification status. However, the level of transcriptional activity in the sperm chromatin of flowering plants has remained unknown. In this study, we observed the distribution of C-terminal domain-phosphorylated RNA polymerase II (RNAPII), which represents a transcriptionally active status in living pollen, and fixed isolated vegetative and sperm nuclei of Arabidopsis thaliana. We found that the transcriptionally active regions were highly limited in sperm chromatin compared with those in vegetative chromatin. RNA sequencing (RNA-seq) analysis showed that the transcription of some RNAPII subunit-encoding genes was highly suppressed in sperm cells, suggesting that the amount of functional RNAPII would be small. In addition, the C-terminal domains of some RNAPII phosphatase-encoding genes, which act as a regulator of the transcription cycle, were actively transcribed in sperm cells. These findings indicate that transcriptional activity is highly suppressed in the sperm chromatin in A. thaliana due to the lack of completely assembled RNAPII and the inhibition of transcriptional elongation of RNAPII by the phosphatase activities.