ß-glucans, SAM, and GSH fluctuations in barley anther tissue culture conditions affect regenerants' DNA methylation and GPRE.

BACKGROUND: Microspore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation-DM, de novo methylation-DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and ß-glucan affect the ability of green plant regeneration efficiency (GPRE). RESULTS: The experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO4: 0.1; 5; 10 µM) and silver salts (AgNO3: 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation. Phenotype variability in ß-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, ß-glucans, Cu(II), GPRE). CONCLUSIONS: We can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE.

Optimizing cannabis cultivation: an efficient in vitro system for flowering induction.

BACKGROUND: Cannabis sativa L. is a versatile medicinal plant known for its therapeutic properties, derived from its diverse array of secondary metabolites synthesized primarily in female flower organs. Breeding cannabis is challenging due to its dioecious nature, strict regulatory requirements, and the need for photoperiod control to trigger flowering, coupled with highly dispersible pollen that can easily contaminate nearby female flowers. This study aimed to develop a protocol for in vitro flowering in cannabis, investigate factors affecting in vitro flower production, and generate viable in vitro seeds, potentially offering a method for producing sterile cannabinoids or advancing breeding techniques. RESULTS: We show that the life cycle of cannabis can be fully completed in tissue culture; plantlets readily produce inflorescences and viable seeds in vitro. Our findings highlight the superior performance of DKW medium with 2% sucrose in a filtered vessel and emphasize the need for low light intensity during flower induction to optimize production. The improved performance in filtered vessels suggests that plants conduct photosynthesis in vitro, highlighting the need for future investigations into the effects of forced ventilation to refine this system. All tested lines readily developed inflorescences upon induction, with a 100% occurrence rate, including male flowering. We revealed the non-dehiscent trait of in vitro anthers, which is advantageous as it allows for multiple crosses to be conducted in vitro without concerns about cross-contamination. CONCLUSION: The current work developed and optimized an effective protocol for in vitro flowering and seed production in cannabis, potentially providing a platform for sterile cannabinoid production and an efficient tool for breeding programs. This system allows for the full and consistent control of plant growth conditions year-round, potentially offering the reliable production of sterile molecules suitable for pharmacological use. As a breeding strategy, this method overcomes the complex challenges of breeding cannabis, such as the need for large facilities, by enabling the production of hundreds of lines in a small facility. By offering precise control over factors such as plant growth regulators, light intensity, photoperiod, and temperature, this system also serves as a valuable tool for studying flowering aspects in cannabis.

Genome-Wide Analysis of the Histone Modification Gene (HM) Family and Expression Investigation during Anther Development in Rice (Oryza sativa L.).

Histone modification plays a crucial role in chromatin remodeling and regulating gene expression, and participates in various biological processes, including plant development and responses to stress. Several gene families related to histone modification have been reported in various plant species. However, the identification of members and their functions in the rice (Oryza sativa L.) histone modification gene family (OsHM) at the whole-genome level remains unclear. In this study, a total of 130 OsHMs were identified through a genome-wide analysis. The OsHM gene family can be classified into 11 subfamilies based on a phylogenetic analysis. An analysis of the genes structures and conserved motifs indicates that members of each subfamily share specific conserved protein structures, suggesting their potential conserved functions. Molecular evolutionary analysis reveals that a significant number of OsHMs proteins originated from gene duplication events, particularly segmental duplications. Additionally, transcriptome analysis demonstrates that OsHMs are widely expressed in various tissues of rice and are responsive to multiple abiotic stresses. Fourteen OsHMs exhibit high expression in rice anthers and peaked at different pollen developmental stages. RT-qPCR results further elucidate the expression patterns of these 14 OsHMs during different developmental stages of anthers, highlighting their high expression during the meiosis and tetrad stages, as well as in the late stage of pollen development. Remarkably, OsSDG713 and OsSDG727 were further identified to be nucleus-localized. This study provides a fundamental framework for further exploring the gene functions of HMs in plants, particularly for researching their functions and potential applications in rice anthers' development and male sterility.

Rapid breeding of an early maturing, high-quality, and high-y.ielding rice cultivar using marker­assisted selection coupled with optimized anther culture.

With the global shift towards healthier eating habits, the focus of the rice industry has evolved from quantity to quality. In China, the Yangtze River Basin is the main area consuming long-grain and high-quality indica rice. Hubei Province, a significant rice-producing area, currently cultivates a limited range of rice varieties, risking degradation and diminishing economic returns. Therefore, it is imperative to cultivate elite rice varieties tailored to the local production conditions and can significantly enhance the added value. This study bred the novel rice cultivar "Runxiangyu", characterized by early maturity, high quality, and high yield. It is a hybrid of Ezhong 5, known for its moderate height and excellent quality, albeit with a long growth period and lack of fragrance, and Yuzhenxiang, renowned for its high quality, short growth period, and fragrance but limited by its tall stature and poor tillering ability. The breeding process utilized optimized anther culture coupled with molecular marker-assisted selection (MAS) and phenotype analysis. In the field, the developed cultivar was 120.9 cm tall and had an entire growth period of 117.5 days, demonstrating moderate disease resistance and excellent heat tolerance. Its grains are fragrant, meeting the national standard of grade two high-quality rice set by the Food Quality Supervision and Inspection Center of the Ministry of Agriculture and Rural Areas). Exhibiting superior agronomic traits, such as plant type, height, growth period, and stress resistance, along with and quality attributes, including grain shape, chalkiness, fragrance, and taste, "Runxiangyu" was certified by the Agricultural Crop Variety Certification Commission of Hubei in 2022. These findings suggested that molecular MAS coupled with optimized anther culture and multi-site phenotype analysis is an efficient and rapid method for crop breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01495-4.

Identification of differentially expressed miRNAs between male sterile and fertile floral buds in watermelon (Citrullus lanatus L.) via high-throughput sequencing.

This experiment used floral buds from watermelon genic male sterile dual-purpose lines as materials to explore the differentially expressed miRNAs (DEMs) between male fertile and sterile floral buds of watermelon. Paraffin sectioning technology was employed for a cytological analysis, and small RNA sequencing was used to explore miRNAs related to anther or pollen development. Cytological analysis indicated that the abnormal development of tapetal cells may cause microspore abortion. Small RNA sequencing identified a total of 314 miRNAs (29 known and 285 novel, which belonged to 12 and 61 miRNA families, respectively) in floral buds. Differential expression revealed 36 (5 known and 31 novel) DEMs between male fertile and sterile buds, 7 and 29 of which were up-regulated and down-regulated, respectively. Target genes analysis showed that the 36 DEMs were predicted to target 577 genes, and these targets might participate in various biological processes, such as response to metal ions, floral organ development, stamen development, anther development, pollen maturation, and programmed cell death. Moreover, pathway analysis indicated that these genes were mainly enriched in purine metabolism, starch and sucrose metabolism, RNA transport, and other pathways. In addition, the 55 miRNA-target modules, including 3 known and 16 novel miRNAs with 30 target genes, might be related to anther or pollen development in watermelon. Our findings provide important miRNA-target modules related to watermelon anther or pollen development and can lay the foundation for biological functional analysis. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04084-6.

Pollinator behaviour and prevalence of the anther smut Antherospora vindobonensis in its host, the Hungarian two-leaf squill (Scilla vindobonensis).

Anther smuts are fungal diseases that have detrimental effects on the biology of their host plants. We investigated the transmission of Anther smut Antherospora vindobonensis to its host, Scilla vindobonensis under natural and laboratory conditions. Plants flowering early in spring were less vulnerable to disease (∼ 20-30% of plants which appeared early in the season were diseased) than flowers that bloom at the end of the season (∼ 60% of plants which appeared late in the season were diseased) which contradicts the patterns observed for other anther smuts species. Diseased plants showed thicker stems, lower nectar production and smaller flower radius than healthy plants. The disease prevalence in S. vindobonensis was not influenced by plant density. The production of spores in diseased flowers was 125 times higher than the production of pollen in healthy flowers. The number of pollen grains attached to pollinator bodies from healthy plants under controlled conditions was similar to the number of spores attached from diseased plants. Experienced honeybees in the field showed almost identical preferences for healthy plants (65%) over diseased ones, as did naïve bumblebees under controlled laboratory conditions (69%). eDNA metabarcoding revealed that healthy flowers attracted a significantly higher number of arthropod species (M = 6.23, SE = 1.48) than diseased flowers (M = 2.32, SE = 0.31). Strong seasonal differences in the risk of infection suggest that S. vindobonensis solves the trade-off between low pollinator availability and low risk of infection at the beginning of the season and high risk of infection and high pollinator availability at the end of season. By reproducing early in the season, plants can reduce the risk of disease.

Callose Deficiency Modulates Plasmodesmata Frequency and Extracellular Distance in Rice Pollen Mother and Tapetal cells.

Fertilization relies on pollen mother cells able to transit from mitosis to meiosis to supply gametes. This process involves remarkable changes at the molecular, cellular and physiological levels including (but not limited to) remodelling of the cell wall. During the meiosis onset, cellulose content at the pollen mother cell walls gradually declines with the concurrent deposition of the polysaccharide callose in anther locules. We aim to understand the biological significance of cellulose-to-callose turnover in pollen mother cells walls using electron microscopic analyses of rice flowers. Our observations indicate that in wild type rice anthers, the mitosis-to-meiosis transition coincides with a gradual reduction in the number of cytoplasmic connections called plasmodesmata. A mutant in the Oryza sativa callose synthase GSL5 (Osgsl5-3), impaired in callose accumulation in premeiotic and meiotic anthers, displayed a greater reduction in plasmodesmata frequency among pollen mother cells and tapetal cells suggesting a role for callose in plasmodesmata maintenance. In addition, a significant increase in extracellular distance between pollen mother cells and impaired premeiotic cell shaping was observed in the Osgsl5-3 mutant. The results suggest that callose-to-cellulose turnover during mitosis-meiosis transition is necessary to maintain cell-to-cell connections and optimal extracellular distance among the central anther locular cells. Findings of this study contribute to our understanding of the regulatory influence of callose metabolism during meiosis initiation in flowering plants.

Overexpression of sesame O-acetylserine(thiol)lyase induces male sterility by delaying tapetum programmed cell death.

Male sterile lines are key resources for hybrid seed production and for ensuring high varietal purity. However, the genes and mechanisms underlying sesame male sterility remain largely unknown. Hence, this study identified an O-acetylserine(thiol)lyase gene SiOASTL1 and functionally characterized its roles in inducing defective anther development. Spatiotemporal expression analysis revealed that SiOASTL1 is significantly (2.7 fold) up-regulated in sterile sesame anthers at the microspore stage compared with fertile ones. Sequence and phylogenetic analyses showed that SiOASTL1 is homologous to Arabidopsis OAS-TL plastid isoforms. We thus overexpressed SiOASTL1 in Arabidopsis to unravel its regulatory roles. Cytological observation revealed that SiOASTL1 overexpression transformed transgenic plants into male sterile lines arising at the microspore development stage. SiOASTL1 overexpression decreased cysteine biosynthesis and down-regulated the expression of the sporopollenin synthesis-related genes, including AtTKPR1, AtTKPR2, AtPKSA, and AtPKSB in transgenic Arabidopsis. Consequently, the tapetum programmed cell death (PCD) was delayed, resulting in the formation of defective pollen grains with irregular walls and empty cytoplasm. Our findings prove that the induction of SiOASTL1 expression disrupts pollen development and contributes to sesame male sterility. Moreover, these results suggest that genetic manipulation of SiOASTL1 expression may facilitate the development of new hybrid varieties in sesame and other crops.

HSP70 and APX1 play important roles in cotton male fertility by mediating ROS homeostasis.

Male sterility is used in the production of hybrid seeds and can improve the breeding efficiency of cotton hybrids. Reactive oxygen species is closely associated with the tapetum and pollen development, but their relationship in cotton male fertility remains unclear. In this study, we comprehensively compared the cytology and proteome of the anthers from an Upland cotton (Gossypium hirsutum) material, Shida 98 (WT), and its nearly-isogenic male sterile line Shida 98A (MS). Cytology indicated delayed PCD in the tapetum and defects in microspores in MS anthers. And further studies revealed disruption of ROS homeostasis. Proteomic analysis identified proteins with differential abundance mainly being related to redox homeostasis, protein folding, and apoptotic signaling pathways. GhAPX1 interacted with GhHSP70 and played a crucial role in the development of cotton anthers. Exogenous application of HSP70 inhibitor increased H2O2 content and decreased the activity of APX1 and pollen viability. The GhAPX1 mutants generated by CRISPR/Cas9-mediated gene editing exhibited premature degradation of the tapetum, significant decrease in pollen viability, and significant increase in H2O2 content. Altogether, our results imply HSP70 and APX1 being the key players jointly regulating male fertility by mediating ROS homeostasis. These results provide insights into the proteins associated with male fertility.

Buzz-pollinating bees deliver thoracic vibrations to flowers through periodic biting.

Pollinator behavior is vital to plant-pollinator interactions, affecting the acquisition of floral rewards, patterns of pollen transfer, and plant reproductive success. During buzz pollination, bees produce vibrations with their indirect flight muscles to extract pollen from tube-like flowers. Vibrations can be transmitted to the flower via the mandibles, abdomen, legs, or thorax directly. Vibration amplitude at the flower determines the rate of pollen release and should vary with the coupling of bee and flower. This coupling often occurs through anther biting, but no studies have quantified how biting affects flower vibration. Here, we used high-speed filmography to investigate how flower vibration amplitude changes during biting in Bombus terrestris visiting two species of buzz-pollinated flowering plants: Solanum dulcamara and Solanum rostratum (Solanaceae). We found that floral buzzing drives head vibrations up to 3 times greater than those of the thorax, which doubles the vibration amplitude of the anther during biting compared with indirect vibration transmission when not biting. However, the efficiency of this vibration transmission depends on the angle at which the bee bites the anther. Variation in transmission mechanisms, combined with the diversity of vibrations across bee species, yields a rich assortment of potential strategies that bees could employ to access rewards from buzz-pollinated flowers.

Anther Culture Protocols for Barley and Wheat.

Doubled haploid (DH) techniques remain valuable tools for wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) genetic improvement, and DH populations are used extensively in breeding and research endeavors. Several techniques are available for DH production in wheat and barley. Here, we describe two simple, robust anther culture methods used to produce more than 15,000 DH wheat and barley lines annually in Australia.

Histochemical and ultrastructural analysis of tapetum and sporoderm development in relation to precocious pollenkitt production of Garcinia dulcis (Roxb.) Kurz.

Garcinia dulcis (Roxb.) Kurz (Clusiaceae) is a medicinal plant native to Southeastern Asia, with a peculiar, precocious pollenkitt production in early microspore development. We aimed to find out whether different secretory activities of the tapetum or a premature sporoderm development provides additional evidence for our recent hypothesis for the precocious pollenkitt production. Histology, histochemistry and ultrastructure of tapetum and sporoderm development during pollenkitt secretion in Garcinia dulcis were conducted, based on light and electron microscopy analysis. The results showed that Garcinia dulcis possesses normal pollen development. The presence of two different pollen coating types, precocious pollenkitt (L1) and common pollenkitt (L2), in the anther tapetum indicate that they are produced in two different active stages of the secretory tapetum. The precocious pollenkitt production and transport to the locule takes place in early active tapetal cells at early tetrad to early microspore stage and is ongoing until late microspore stage. The production of the second type of pollenkitt (L2) starts shortly after the first active tapetum stage together with the formation of sporopollenin precursors. The sporoderm formation was completed at late microspore stage, when the tapetal cell walls start to disintegrate. Orbicules are lining the inner tapetum wall at middle to late microspore stage. ER (during early microspore stage) and plastids (during late microspore stage) were the two main sources of pollenkitt, which finally fused to pollenkitt droplets when the tapetal cells degenerated at mature bicellular pollen stage.

An Introduction to Plant Cell, Tissue, and Organ Culture: Current Status and Perspectives.

Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.

Closed floral structure for self-pollination in cultivated tomato.

Cultivated tomatoes exhibit cleistogamy - self-pollination within closed flowers. Wu et al. report that three HD-Zip IV genes and Style2.1 coordinately control anther trichome formation and style length to form closed anther cones that underpin the development of cleistogamy. Further exploration of causal variation and regulatory elements could provide targets for plant breeding.

The regulatory mechanism of rapid lignification for timely anther dehiscence.

Anther dehiscence is a crucial event in plant reproduction, tightly regulated and dependent on the lignification of the anther endothecium. In this study, we investigated the rapid lignification process that ensures timely anther dehiscence in Arabidopsis. Our findings reveal that endothecium lignification can be divided into two distinct phases. During Phase I, lignin precursors are synthesized without polymerization, while Phase II involves simultaneous synthesis of lignin precursors and polymerization. The transcription factors MYB26, NST1/2, and ARF17 specifically regulate the pathway responsible for the synthesis and polymerization of lignin monomers in Phase II. MYB26-NST1/2 is the key regulatory pathway responsible for endothecium lignification, while ARF17 facilitates this process by interacting with MYB26. Interestingly, our results demonstrate that the lignification of the endothecium, which occurs within approximately 26 h, is much faster than that of the vascular tissue. These findings provide valuable insights into the regulation mechanism of rapid lignification in the endothecium, which enables timely anther dehiscence and successful pollen release during plant reproduction.

Rice Big Grain1 improves grain yield in ectopically expressing rice and heterologously expressing tobacco plants.

Functional genomics through transgenesis has provided faster and more reliable methods for identifying, characterizing, and utilizing genes or quantitative trait loci linked to agronomic traits to target yield. The present study explored the role of Big Grain1 (BG1) gene of rice (Oryza sativa L.) in yield improvement of crop plants. We aimed to identify the genetic variation of OsBG1 in various indica rice cultivars by studying the allelic polymorphism of the gene, while also investigating the gene's potential to increase crop yield through the transgenic approach. Our study reports the presence of an extra 393 bp sequence having two 6 bp enhancer elements in the 3' regulatory sequence of OsBG1 in the large-grain cultivar IR64 but not in the small-grain cultivar Badshahbhog. A single copy of the OsBG1 gene in both the cultivars and a 4.1-fold higher expression of OsBG1 in IR64 than in Badshahbhog imply that the grain size is positively correlated with the level of OsBG1 expression in rice. The ectopic expression of OsBG1 under the endosperm-specific glutelin C promoter in Badshahbhog enhanced the flag leaf length, panicle weight, and panicle length by an average of 33.2%, 33.7%, and 30.5%, respectively. The length of anthers, spikelet fertility, and grain yield per plant increased in transgenic rice lines by an average of 27.5%, 8.3%, and 54.4%, respectively. Heterologous expression of OsBG1 under the constitutive 2xCaMV35S promoter improved the number of seed pods per plant and seed yield per plant in transgenic tobacco lines by an average of 2.2-fold and 2.6-fold, respectively. Improving crop yield is crucial to ensure food security and socio-economic stability, and identifying suitable genetic factor is the essential step towards this endeavor. Our findings suggest that the OsBG1 gene is a promising candidate for improving the grain yield of monocot and dicot plant systems by molecular breeding and genetic engineering.

Identification of anther thermotolerance genes by the integration of linkage and association analysis in maize.

Maize is one of the world's most important staple crops, yet its production is increasingly threatened by the rising frequency of high-temperature stress (HTS). To investigate the genetic basis of anther thermotolerance under field conditions, we performed linkage and association analysis to identify HTS response quantitative trait loci (QTL) using three recombinant inbred line (RIL) populations and an association panel containing 375 diverse maize inbred lines. These analyses resulted in the identification of 16 co-located large QTL intervals. Among the 37 candidate genes identified in these QTL intervals, five have rice or Arabidopsis homologs known to influence pollen and filament development. Notably, one of the candidate genes, ZmDUP707, has been subject to selection pressure during breeding. Its expression is suppressed by HTS, leading to pollen abortion and barren seeds. We also identified several additional candidate genes potentially underly QTL previously reported by other researchers. Taken together, our results provide a pool of valuable candidate genes that could be employed by future breeding programs aiming at enhancing maize HTS tolerance.

Tetraploid interspecific hybrids between Asian and African rice species restore fertility depending on killer-protector loci for hybrid sterility.

Interspecific F1 hybrids between Asian (Oryza sativa) and African rice (Oryza glaberrima) exhibit severe sterility caused by the accumulation of hybrid sterility genes/loci at 15 or more loci. The mechanisms underlying the hybrid sterility genes are largely unknown; however, a few genes associated with the killer-protector system, which is the system most frequently associated with hybrid sterility genes, have been identified. We previously produced fertile plants as tetraploids derived from diploid interspecific F1 hybrids through anther culture; therefore, it was suggested that hybrid sterility could be overcome following tetraploidization. We investigated whether tetraploid interspecific plants produced by crossing are fertile and tested the involvement of hybrid sterility genes in the process. Fertile tetraploid interspecific F1 hybrid plants were obtained by crossing 2 tetraploids of O. sativa and O. glaberrima. To elucidate the relationships between pollen fertility and the hybrid sterility loci in the tetraploid F1 microspores, we performed genetic analyses of the tetraploid F2 hybrids and diploid plants obtained from the microspores of tetraploid interspecific hybrids by anther culture. The result suggested that the tetraploid interspecific hybrids overcame pollen and seed infertility based on the proportion of loci with the killer-protector system present in the tetraploids. The heterozygous hybrid sterility loci with the killer-protector system in the tetraploid segregate the homozygous killed allele (16.7-21.4%), with more than three-quarters of the gametes surviving. We theoretically and experimentally demonstrated that fertile rice progenies can be grown from tetraploid interspecific hybrids.

Exploring the potential of Paris polyphylla var. yunnanensis pollen manipulation in modifying seed dormancy.

Paris polyphylla var. yunnanensis, a well-known Chinese medicinal herb, shows a unique physiological trait characterized by the cyclic opening and closing of its anthers after pollen maturation. The aim of this study was to explore the implications of this phenomenon on breeding. RNA sequencing coupled with methylation sequencing was used to scrutinize and compare gene expression profiles and methylation alterations in pollen and seeds during anther opening and closing, along with cold exposure. Genes enriched within Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were examined to identify gene clusters susceptible to temperature-related methylation changes in both pollen and seeds. Four pollen treatment models, namely, normal control, "pollen protected from low temperatures," "pollen from just-opened anther," and "pollen from close-blocked anther," were used to produce corresponding seeds via artificial pollination. Subsequently, qRT-PCR was used to validate modifications in the expression patterns of marker genes in pollinated seeds under diverse treatment scenarios. Genes exhibiting significant differences in expression between anthers and normal tissues, along with gene regions linked to methylation variations attributed to low-temperature-treated pollen and seeds, were identified through transcriptomic analysis. Convergence was observed in three signaling pathways: oxidative phosphorylation (ko00190), plant hormone signal transduction (Ko04075), and zeatin biosynthesis (ko00908). Notably, gene clusters prone to temperature-induced methylation changes, such as NADH-ubiquinone oxidoreductase chain 5, plasma membrane ATPase 4, cytochrome c oxidase subunit 2, cis-zeatin O-glucosyltransferase, ABSCISIC ACID-INSENSITIVE 5-like protein 4, and indole-3-acetic acid-amido synthetase (IAAS), were identified. Evaluation using various pollen pollination models revealed altered expression patterns of five dormancy-regulating marker genes: IAAS, sucrose synthase (SUS), gibberellin 2-oxidase (GA2ox), ABA INSENSITIVE 2 (ABI2), and auxin-repressed protein (ARP), in seeds pollinated with pollen from close-blocked anthers, cold-protected pollen, and pollen from freshly opened anthers. The close-blocked anther treatment led to significantly upregulated expression of IAAS, SUS, GA2ox, and ABI2, whereas ARP expression decreased markedly, indicating a propensity toward prolonged seed dormancy. Conversely, in the low-temperature-protected anther model, SUS, ARP, GA2ox, and IAAS exhibited reduced expression levels, whereas the expression of ABI2 was upregulated, overall facilitating seed germination.

Genome-Wide Identification and Expression Analysis of the PHD Finger Gene Family in Pea (Pisum sativum).

The plant homeodomain finger (PHD finger) protein, a type of zinc finger protein extensively distributed in eukaryotes, plays diverse roles in regulating plant growth and development. While PHD finger proteins have been identified in various species, their functions remain largely unexplored in pea (Pisum sativum). In this study, we identified 84 members of the PHD finger gene family in pea, which displayed an uneven distribution across seven chromosomes. Through a comprehensive analysis using data from Arabidopsis thaliana and Medicago truncatula, we categorized the PHD finger proteins into 20 subfamilies via phylogenetic tree analysis. Each subfamily exhibited distinct variations in terms of quantity, genetic structure, conserved domains, and physical and chemical properties. Collinearity analysis revealed conserved evolutionary relationships among the PHD finger genes across the three different species. Furthermore, we identified the conserved and important roles of the subfamily M members in anther development. RT-qPCR and in situ hybridization revealed high expression of the pea subfamily M members PsPHD11 and PsPHD16 in microspores and the tapetum layer. In conclusion, this analysis of the PHD finger family in pea provides valuable guidance for future research on the biological roles of PHD finger proteins in pea and other leguminous plants.