Genomic analysis of a spontaneous unifoliate mutant reveals gene candidates associated with compound leaf development in Vigna unguiculata [L] Walp.

Molecular mechanisms which underpin compound leaf development in some legumes have been reported, but there is no previous study on the molecular genetic control of compound leaf formation in Vigna unguiculata (cowpea), an important dryland legume of African origin. In most studied species with compound leaves, class 1 KNOTTED-LIKE HOMEOBOX genes expressed in developing leaf primordia sustain morphogenetic activity, allowing leaf dissection and the development of leaflets. Other genes, such as, SINGLE LEAFLET1 in Medicago truncatula and Trifoliate in Solanum lycopersicum, are also implicated in regulating compound leaf patterning. To set the pace for an in-depth understanding of the genetics of compound leaf development in cowpea, we applied RNA-seq and whole genome shotgun sequence datasets of a spontaneous cowpea unifoliate mutant and its trifoliate wild-type cultivar to conduct comparative reference-based gene expression, de novo genome-wide isoform switch, and genome variant analyses between the two genotypes. Our results suggest that genomic variants upstream of LATE ELONGATED HYPOCOTYL and down-stream of REVEILLE4, BRASSINOSTERIOD INSENSITIVE1 and LATERAL ORGAN BOUNDARIES result in down-regulation of key components of cowpea circadian rhythm central oscillator and brassinosteroid signaling, resulting in unifoliate leaves and brassinosteroid-deficient-like phenotypes. We have stated hypotheses that will guide follow-up studies expected to provide more insights.

Prognostic analysis of radiation-induced liver damage following carbon-ion radiotherapy for hepatocellular carcinoma.

BACKGROUND: Radiation-induced liver damage (RILD) occasionally occurs following carbon-ion radiotherapy (CIRT) for liver tumors, such as hepatocellular carcinoma (HCC), in patients with impaired liver function disease. However, the associated risk factors remain unknown. The present study aimed to determine the risk factors of RILD after CIRT. METHODS: We retrospectively analyzed 108 patients with HCC treated with CIRT at the Osaka Heavy Ion Therapy Center between December 2018 and December 2022. RILD was defined as a worsening of two or more points in the Child-Pugh score within 12 months following CIRT. The median age of the patients was 76 years (range 47-95 years), and the median tumor diameter was 41 mm (range 5-160 mm). Based on the pretreatment liver function, 98 and 10 patients were categorized as Child-Pugh class A and B, respectively. We analyzed patients who received a radiation dose of 60 Gy (relative biological effectiveness [RBE]) in four fractions. The median follow-up period was 9.7 months (range 2.3-41.1 months), and RILD was observed in 11 patients (10.1%). RESULTS: Multivariate analysis showed that pretreatment Child-Pugh score B (p = 0.003, hazard ratio [HR] = 6.90) and normal liver volume spared from < 30 Gy RBE (VS30 < 739 cm3) (p = 0.009, HR = 5.22) were significant risk factors for RILD. The one-year cumulative incidences of RILD stratified by Child-Pugh class A or B and VS30 < 739 cm3 or ≥ 739 cm3 were 10.3% or 51.8% and 39.6% or 9.2%, respectively. CONCLUSION: In conclusion, the pretreatment Child-Pugh score and VS30 of the liver are significant risk factors for RILD following CIRT for HCC.

Genome and transcriptome analyses reveal genes involved in the formation of fine ridges on petal epidermal cells in Hibiscus trionum.

Hibiscus trionum, commonly known as the 'Flower of an Hour', is an easily cultivated plant in the Malvaceae family that is widespread in tropical and temperate regions, including drylands. The purple base part of its petal exhibits structural colour due to the fine ridges on the epidermal cell surface, and the molecular mechanism of ridge formation has been actively investigated. We performed genome sequencing of H. trionum using a long-read sequencing technology with transcriptome and pathway analyses to identify candidate genes for fine structure formation. The ortholog of AtSHINE1, which is involved in the biosynthesis of cuticular wax in Arabidopsis thaliana, was significantly overexpressed in the iridescent tissue. In addition, orthologs of AtCUS2 and AtCYP77A, which contribute to cutin synthesis, were also overexpressed. Our results provide important insights into the formation of fine ridges on epidermal cells in plants using H. trionum as a model.

Metabolic and transcriptomic profiling during wheat seed development under progressive drought conditions.

Globally, bread wheat (Triticum aestivum) is one of the most important staple foods; when exposed to drought, wheat yields decline. Although much research has been performed to generate higher yield wheat cultivars, there have been few studies on improving end-product quality under drought stress, even though wheat is processed into flour to produce so many foods, such as bread, noodles, pancakes, cakes, and cookies. Recently, wheat cultivation has been affected by severe drought caused by global climate change. In previous studies, seed shrinkage was observed in wheat exposed to continuous drought stress during seed development. In this study, we investigated how progressive drought stress affected seed development by metabolomic and transcriptomic analyses. Metabolite profiling revealed the drought-sensitive line reduced accumulation of proline and sugar compared with the water-saving, drought-tolerant transgenic line overexpressing the abscisic acid receptor TaPYL4 under drought conditions in spikelets with developing seeds. Meanwhile, the expressions of genes involved in translation, starch biosynthesis, and proline and arginine biosynthesis was downregulated in the drought-sensitive line. These findings suggest that seed shrinkage, exemplifying a deficiency in endosperm, arose from the hindered biosynthesis of crucial components including seed storage proteins, starch, amino acids, and sugars, ultimately leading to their inadequate accumulation within spikelets. Water-saving drought tolerant traits of wheat would aid in supporting seed formation under drought conditions.

CRISPR-FISH: A CRISPR/Cas9-Based In Situ Labeling Method.

Fluorescence in situ hybridization (FISH) has been widely used to visualize target DNA sequences in fixed chromosome samples by denaturing the dsDNA to allow complementary probe hybridization, thus damaging the chromatin structure by harsh treatments. To overcome this limitation, a CRISPR/Cas9-based in situ labeling method was developed, termed CRISPR-FISH. This method is also known as RNA-guided endonuclease-in situ labeling (RGEN-ISL). Here we present different protocols for the application of CRISPR-FISH on acetic acid: ethanol or formaldehyde-fixed nuclei and chromosomes as well as tissue sections for labeling repetitive sequences in a range of plant species. In addition, methods on how immunostaining can be combined with CRISPR-FISH are provided.

Holocentromeres can consist of merely a few megabase-sized satellite arrays.

The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.

Chemical emasculation in cowpea (Vigna unguiculata (L.) Walp.) and dicotyledonous model species using trifluoromethanesulfonamide (TFMSA).

Hybridization plays an indispensable role in creating the diversity associated with plant evolution and genetic improvement of crops. Production of hybrids requires control of pollination and avoidance of self-pollination for species that are predominantly autogamous. Hand emasculation, male sterility genes or male gametocides have been used in several plant species to induce pollen sterility. However, in cowpea (Vigna unguiculata (L.) Walp), a self-pollinated cleistogamous dryland crop, only hand emasculation is used, but it is tedious and time-consuming. In this study, male sterility was effectively induced in cowpea and two dicotyledonous model species (Arabidopsis thaliana (L.) Heynh. and Nicotiana benthamiana Domin) using trifluoromethanesulfonamide (TFMSA). Pollen viability assays using Alexander staining showed that 30 ml of 1000 mg/l TFMSA with two-time treatments of one-week interval at the early stage of the reproductive phase under field or greenhouse conditions induced 99% pollen sterility in cowpea. TFMSA treatment induced non-functional pollen in diploid A. thaliana at two-time treatment of 10 ml of 125-250 mg/l per plant and N. benthamiana at two-time treatment of 10 ml of 250-1000 mg/l per plant. TFMSA-treated cowpea plants produced hybrid seeds when used as the female parent in crosses with non-treated plants used as male parents, suggesting that TFMSA had no effect on female functionality in cowpea. The ease of TFMSA treatment and its effectiveness to induce pollen sterility in a wide range of cowpea genotypes, and in the two model plant species tested in this study, may expand the scope of techniques for rapid pollination control in self-pollinated species, with potential applications in plant breeding and plant reproduction science.

Cowpea speed breeding using regulated growth chamber conditions and seeds of oven-dried immature pods potentially accommodates eight generations per year.

BACKGROUND: Cowpea is a dryland crop with potential to improve food security in sub-Saharan Africa, where it is mostly produced and consumed. Contemporary plant improvement technologies, including genome editing, marker-assisted selection, and optimized transformation protocols, are being deployed to improve cowpea characteristics. Integrating speed breeding with these technologies would accelerate genetic gain in cowpea breeding. There are established speed breeding protocols for other important legumes, such as soybean, peanut, and chickpea, but none has been previously reported for cowpea. RESULTS: With the aid of regulated growth conditions in two different chamber types, as well as the cultivation of new plant generations from seeds of oven-dried immature pods, we developed and validated, for the first time, an efficient speed breeding protocol that accommodates approximately seven to eight breeding generations per year for 3 cowpea genotypes. The 3 cowpea genotypes were evaluated under controlled growth conditions in light-emitting diode and metal halide lamp chambers to determine the effect of CO2 supplementation on flowering and maturation durations, optimum conditions for plant growth, cross pollination, and pod development. Elevated CO2 concentration had no influence on either flowering time or pod development. Adequate temperature, relative humidity and light intensity improved plant development and the rate of successful hand pollination, and  cultivating seeds of 11-day-old immature pods oven-dried at 39 °C for 2 days resulted in at least a 62% reduction in the time between pollination and sowing of the next plant generation. The plants cultivated from seeds of the oven-dried immature pods showed no defect at any stage of development. CONCLUSIONS: Using the speed breeding protocol developed in this study, cowpea breeding cycles can be increased from the traditional one cycle per year in the field to as many as 8 generations per year in regulated growth chamber conditions. This protocol has no special technical requirements; hence, it can be implemented in any standard growth chamber. This would fast-track development, testing, validation, and utilization of improved cowpea cultivars.

The Evolutionary Dynamics of Repetitive DNA and Its Impact on the Genome Diversification in the Genus Sorghum.

Polyploidization is an evolutionary event leading to structural changes of the genome(s), particularly allopolyploidization, which combines different genomes of distinct species. The tetraploid species, Sorghum halepense, is assumed an allopolyploid species formed by hybridization between diploid S. bicolor and S. propinquum. The repeat profiles of S. bicolor, S. halepense, and their relatives were compared to elucidate the repeats' role in shaping their genomes. The repeat frequencies and profiles of the three diploid accessions (S. bicolor, S. bicolor ssp. verticilliflorum, and S. bicolor var. technicum) and two tetraploid accessions (S. halepense) are similar. However, the polymorphic distribution of the subtelomeric satellites preferentially enriched in the tetraploid S. halepense indicates drastic genome rearrangements after the allopolyploidization event. Verified by CENH3 chromatin immunoprecipitation (ChIP)-sequencing and fluorescence in situ hybridization (FISH) analysis the centromeres of S. bicolor are mainly composed of the abundant satellite SorSat137 (CEN38) and diverse CRMs, Athila of Ty3_gypsy and Ty1_copia-SIRE long terminal repeat (LTR) retroelements. A similar centromere composition was found in S. halepense. The potential contribution of S. bicolor in the formation of tetraploid S. halepense is discussed.

Development and regeneration of wheat-rice hybrid zygotes produced by in vitro fertilization system.

Hybridization plays a decisive role in the evolution and diversification of angiosperms. However, the mechanisms of wide hybridization remain open because pre- and post-fertilization barriers limit the production and development of inter-subfamily/intergeneric zygotes, respectively. We examined hybridization between wheat and rice using an in vitro fertilization (IVF) system to bypass these barriers. Several gamete combinations of allopolyploid wheat-rice hybrid zygotes were successfully produced, and the developmental profiles of hybrid zygotes were analyzed. Hybrid zygotes derived from one rice egg cell and one wheat sperm cell ceased at the multicellular embryo-like structure stage. This developmental barrier was overcome by adding one wheat egg cell to the wheat-rice hybrid zygote. In the reciprocal combination, one wheat egg and one rice sperm cell, the resulting hybrid zygotes failed to divide. However, doubling the dosage of rice sperm cell allowed the hybrid zygotes to develop into plantlets. Rice chromosomes appeared to be progressively eliminated during the early developmental stage of these hybrid embryos, and c. 20% of regenerated plants showed abnormal morphology. These results suggest that hybrid breakdown can be overcome through optimization of gamete combinations, and the present hybrid will provide a new horizon for utilization of inter-subfamily genetic resources.

Unequal contribution of two paralogous CENH3 variants in cowpea centromere function.

In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the  diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.

Heat Sensing and Lipid Reprograming as a Signaling Switch for Heat Stress Responses in Wheat.

Temperature is an essential physical factor that affects the plant life cycle. Almost all plant species have evolved a robust signal transduction system that enables them to sense changes in the surrounding temperature, relay this message and accordingly adjust their metabolism and cellular functions to avoid heat stress-related damage. Wheat (Triticum aestivum), being a cool-season crop, is very sensitive to heat stress. Any increase in the ambient temperature, especially at the reproductive and grain-filling stages, can cause a drastic loss in wheat yield. Heat stress causes lipid peroxidation due to oxidative stress, resulting in the damage of thylakoid membranes and the disruption of their function, which ultimately decreases photosynthesis and crop yield. The cell membrane/plasma membrane plays prominent roles as an interface system that perceives and translates the changes in environmental signals into intracellular responses. Thus, membrane lipid composition is a critical factor in heat stress tolerance or susceptibility in wheat. In this review, we elucidate the possible involvement of calcium influx as an early heat stress-responsive mechanism in wheat plants. In addition, the physiological implications underlying the changes in lipid metabolism under high-temperature stress in wheat and other plant species will be discussed. In-depth knowledge about wheat lipid reprograming can help develop heat-tolerant wheat varieties and provide approaches to solve the impact of global climate change.

Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells.

The 3D organization of chromatin plays an important role in genome stability and many other pivotal biological programs. Therefore, the establishment of imaging methods, which enable us to study the dynamics of chromatin in living cells, is necessary. Although primary live cell imaging methods were a breakthrough, there is a need to develop more specific labeling techniques. With the discovery of programmable DNA binding proteins, such zinc finger proteins (ZFP), transcription activator-like effectors (TALE), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a major leap forward was made. Here, we review the applications and potential of fluorescent repressor-operator systems, programmable DNA binding proteins with an emphasis on CRISPR-based chromatin imaging in living and fixed cells, and their potential application in plant science.

CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication.

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.

RNA-guided endonuclease - in situ labelling (RGEN-ISL): a fast CRISPR/Cas9-based method to label genomic sequences in various species.

Visualising the spatio-temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two-part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN-ISL (RNA-guided endonuclease - in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans-activating crRNAs allows the multiplexing of RGEN-ISL. Moreover, this technique is combinable with immunohistochemistry. Real-time visualisation of the CRISPR/Cas9-mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN-ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology.

Evaluation of the accuracy and clinical practicality of a calculation system for patient positional displacement in carbon ion radiotherapy at five sites.

PURPOSE: We developed a system for calculating patient positional displacement between digital radiography images (DRs) and digitally reconstructed radiography images (DRRs) to reduce patient radiation exposure, minimize individual differences between radiological technologists in patient positioning, and decrease positioning time. The accuracy of this system at five sites was evaluated with clinical data from cancer patients. The dependence of calculation accuracy on the size of the region of interest (ROI) and initial position was evaluated for clinical use. METHODS: For a preliminary verification, treatment planning and positioning data from eight setup patterns using a head and neck phantom were evaluated. Following this, data from 50 patients with prostate, lung, head and neck, liver, or pancreatic cancer (n = 10 each) were evaluated. Root mean square errors (RMSEs) between the results calculated by our system and the reference positions were assessed. The reference positions were manually determined by two radiological technologists to best-matching positions with orthogonal DRs and DRRs in six axial directions. The ROI size dependence was evaluated by comparing RMSEs for three different ROI sizes. Additionally, dependence on initial position parameters was evaluated by comparing RMSEs for four position patterns. RESULTS: For the phantom study, the average (± standard deviation) translation error was 0.17 ± 0.05, rotation error was 0.17 ± 0.07, and ΔD was 0.14 ± 0.05. Using the optimal ROI size for each patient site, all cases of prostate, lung, and head and neck cancer with initial position parameters of 10 mm or under were acceptable in our tolerance. However, only four liver cancer cases and three pancreatic cancer cases were acceptable, because of low-reproducibility regions in the ROIs. CONCLUSION: Our system has clinical practicality for prostate, lung, and head and neck cancer cases. Additionally, our findings suggest ROI size dependence in some cases.

A histological examination of spinal reconstruction using a frozen bone autograft.

Our aim was to compare the process of bone formation after reconstruction of the vertebral body using a titanium cage with either a liquid nitrogen-treated (frozen) bone autograft or non-treated fresh bone autograft. Twelve canine beagles underwent anterior reconstruction of the 5th lumbar vertebrae using a titanium cage and bone autograft. Bone formation was compared across four experimental groups: fresh bone autograft groups, with animals sacrificed at either 8 or 16 weeks post-reconstruction, and liquid nitrogen-treated (frozen) bone autograft groups, with animals again sacrificed at either 8 or 16 weeks post-reconstruction. Bone formation was evaluated histologically by calculating the proportion of 'reaction' and 'mature bone' regions at the ends of the cage, its center, and ventral/dorsal sides. The reaction region contained osteocytes with a nucleus and osteoblasts accumulated on the surface of an osteoid, while a laminar structure was visible for mature bone regions. For fresh bone autografts, the reaction and mature bone regions significantly increased from 8 to 16 weeks post-reconstruction. By comparison, for frozen autografts, the reaction bone region did not significantly increase from 8 to 16 weeks post-reconstruction, while the mature bone region did increase over this time period. The proportion of reaction bone was higher at the ends and dorsal side of the cage at 8 weeks, for both graft types, with greater bone formation at the center of the cage at 16 weeks only for the fresh bone autograft. Therefore, bone formation in the anterior spinal reconstruction site tended to be delayed when using a frozen bone autograft compared to a fresh bone autograft. The bone formation process, however, was similar for both groups, beginning at the ends and dorsal side of the cage adjacent to the surrounding vertebral bone.

Centromere location in Arabidopsis is unaltered by extreme divergence in CENH3 protein sequence.

During cell division, spindle fibers attach to chromosomes at centromeres. The DNA sequence at regional centromeres is fast evolving with no conserved genetic signature for centromere identity. Instead CENH3, a centromere-specific histone H3 variant, is the epigenetic signature that specifies centromere location across both plant and animal kingdoms. Paradoxically, CENH3 is also adaptively evolving. An ongoing question is whether CENH3 evolution is driven by a functional relationship with the underlying DNA sequence. Here, we demonstrate that despite extensive protein sequence divergence, CENH3 histones from distant species assemble centromeres on the same underlying DNA sequence. We first characterized the organization and diversity of centromere repeats in wild-type Arabidopsis thaliana We show that A. thaliana CENH3-containing nucleosomes exhibit a strong preference for a unique subset of centromeric repeats. These sequences are largely missing from the genome assemblies and represent the youngest and most homogeneous class of repeats. Next, we tested the evolutionary specificity of this interaction in a background in which the native A. thaliana CENH3 is replaced with CENH3s from distant species. Strikingly, we find that CENH3 from Lepidium oleraceum and Zea mays, although specifying epigenetically weaker centromeres that result in genome elimination upon outcrossing, show a binding pattern on A. thaliana centromere repeats that is indistinguishable from the native CENH3. Our results demonstrate positional stability of a highly diverged CENH3 on independently evolved repeats, suggesting that the sequence specificity of centromeres is determined by a mechanism independent of CENH3.

Wide Hybridization Between Oat and Pearl Millet.

Wide hybridization is a one of the important techniques in plant breeding. Oat (Avena sativa L.) and pearl millet (Pennisetum glaucum L.) belong to different subfamilies of Poaceae. In generally, such distant relative species show uniparental chromosome elimination after successful fertilization. However, all seven pearl millet chromosomes are retained beside the genome of oat during embryogenesis. Hybrid seedlings develop, but show necrosis after light irradiation. Here, a detailed protocol for wide hybridization between oat and pearl millet is described.

[Three-Dimensional Dosimetry of Carbon Ion Beams by Gel Dosimeters].

A three-dimensional dosimetry method is strongly required in the dose distribution measurement of a patient QA of a heavy ion therapy. Nanocomposite Fricke gel dosimeters are the most possible candidate for this purpose. Experimental dose distribution measurements were carried out using a scanning irradiation port of Gunma University Heavy Ion Medical Center. The result showed no significant LET dependence and indicated a possibility for a precise dosimetry of a heavy ion therapy. It also indicated the importance of three-dimensional dosimetry in the commissioning process of the treatment accelerator.