A robust transformer-based pipeline of 3D cell alignment, denoise and instance segmentation on electron microscopy sequence images.

Germline cells are critical for transmitting genetic information to subsequent generations in biological organisms. While their differentiation from somatic cells during embryonic development is well-documented in most animals, the regulatory mechanisms initiating plant germline cells are not well understood. To thoroughly investigate the complex morphological transformations of their ultrastructure over developmental time, nanoscale 3D reconstruction of entire plant tissues is necessary, achievable exclusively through electron microscopy imaging. This paper presents a full-process framework designed for reconstructing large-volume plant tissue from serial electron microscopy images. The framework ensures end-to-end direct output of reconstruction results, including topological networks and morphological analysis. The proposed 3D cell alignment, denoise, and instance segmentation pipeline (3DCADS) leverages deep learning to provide a cell instance segmentation workflow for electron microscopy image series, ensuring accurate and robust 3D cell reconstructions with high computational efficiency. The pipeline involves five stages: the registration of electron microscopy serial images; image enhancement and denoising; semantic segmentation using a Transformer-based neural network; instance segmentation through a supervoxel-based clustering algorithm; and an automated analysis and statistical assessment of the reconstruction results, with the mapping of topological connections. The 3DCADS model's precision was validated on a plant tissue ground-truth dataset, outperforming traditional baseline models and deep learning baselines in overall accuracy. The framework was applied to the reconstruction of early meiosis stages in the anthers of Arabidopsis thaliana, resulting in a topological connectivity network and analysis of morphological parameters and characteristics of cell distribution. The experiment underscores the 3DCADS model's potential for biological tissue identification and its significance in quantitative analysis of plant cell development, crucial for examining samples across different genetic phenotypes and mutations in plant development. Additionally, the paper discusses the regulatory mechanisms of Arabidopsis thaliana's germline cells and the development of stamen cells before meiosis, offering new insights into the transition from somatic to germline cell fate in plants.

Plant-on-chip: Core morphogenesis processes in the tiny plant Wolffia australiana.

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the minimalist known angiosperm to overcome this challenge and provide a model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the plant-on-chip culture system and demonstrate the application of advanced technologies such as single-nucleus RNA-sequencing, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can decipher the core regulatory mechanisms of plant morphogenesis.

OsMADS58 Stabilizes Gene Regulatory Circuits during Rice Stamen Development.

Rice (Oryza sativa) OsMADS58 is a C-class MADS box protein, and characterization of a transposon insertion mutant osmads58 suggested that OsMADS58 plays a role in stamen development. However, as no null mutation has been obtained, its role has remained unclear. Here, we report that the CRISPR knockout mutant osmads58 exhibits complex altered phenotypes, including anomalous diploid germ cells, aberrant meiosis, and delayed tapetum degeneration. This CRISPR mutant line exhibited stronger changes in expression of OsMADS58 target genes compared with the osmads58 dSpm (transposon insertion) line, along with changes in multiple pathways related to early stamen development. Notably, transcriptional regulatory circuits in young panicles covering the stamen at stages 4-6 were substantially altered in the CRISPR line compared to the dSpm line. These findings strongly suggest that the pleiotropic effects of OsMADS58 on stamen development derive from a potential role in stabilizing gene regulatory circuits during early stamen development. Thus, this work opens new avenues for viewing and deciphering the regulatory mechanisms of early stamen development from a network perspective.

The genome of homosporous maidenhair fern sheds light on the euphyllophyte evolution and defences.

Euphyllophytes encompass almost all extant plants, including two sister clades, ferns and seed plants. Decoding genomes of ferns is the key to deep insight into the origin of euphyllophytes and the evolution of seed plants. Here we report a chromosome-level genome assembly of Adiantum capillus-veneris L., a model homosporous fern. This fern genome comprises 30 pseudochromosomes with a size of 4.8-gigabase and a contig N50 length of 16.22 Mb. Gene co-expression network analysis uncovered that homospore development in ferns has relatively high genetic similarities with that of the pollen in seed plants. Analysing fern defence response expands understanding of evolution and diversity in endogenous bioactive jasmonates in plants. Moreover, comparing fern genomes with those of other land plants reveals changes in gene families important for the evolutionary novelties within the euphyllophyte clade. These results lay a foundation for studies on fern genome evolution and function, as well as the origin and evolution of euphyllophytes.

Origins of the seed: The "golden-trio hypothesis".

The seed is an evolutionary innovation in the plant kingdom. While human civilization depends heavily on seed production, how the seed trait emerged remains elusive. In this opinion article, a "golden-trio hypothesis" is proposed based on our investigations of LEC1 gene functions in Adiantum capillus-veneris. This hypothesis posits that a "seed program" arose from spatiotemporal integration of three key components: assimilate flow, ABA-mediated stress responses, and stress-induced LEC1 expression. Thus, the evolutionary innovation of seeds should be considered not a simple event resulting from new genes; rather, it represents the outcome of a series of physiological and morphological innovations that emerged prior to and regardless of the origin of the seed program. This new perspective could help us tackle some long-standing questions around the puzzling origin of seeds.

Rice Cell Division Cycle 20s are required for faithful chromosome segregation and cytokinesis during meiosis.

Chromosome segregation must be under strict regulation to maintain chromosome euploidy and stability. Cell Division Cycle 20 (CDC20) is an essential cell cycle regulator that promotes the metaphase-to-anaphase transition and functions in the spindle assembly checkpoint, a surveillance pathway that ensures the fidelity of chromosome segregation. Plant CDC20 genes are present in multiple copies, and whether CDC20s have the same functions in plants as in yeast and animals is unclear, given the potential for divergence or redundancy among the multiple copies. Here, we studied all three CDC20 genes in rice (Oryza sativa) and constructed two triple mutants by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated genome editing to explore their roles in development. Knocking out all three CDC20 genes led to total sterility but did not affect vegetative development. Loss of the three CDC20 proteins did not alter mitotic division but severely disrupted meiosis as a result of asynchronous and unequal chromosome segregation, chromosome lagging, and premature separation of chromatids. Immunofluorescence of tubulin revealed malformed meiotic spindles in microsporocytes of the triple mutants. Furthermore, cytokinesis of meiosis I was absent or abnormal, and cytokinesis II was completely prevented in all mutant microsporocytes; thus, no tetrads or pollen formed in either cdc20 triple mutant. Finally, the subcellular structures and functions of the tapetum were disturbed by the lack of CDC20 proteins. These findings demonstrate that the three rice CDC20s play redundant roles but are indispensable for faithful meiotic chromosome segregation and cytokinesis, which are required for the production of fertile microspores.

Auxin guides germ-cell specification in Arabidopsis anthers.

Germ cells (GCs) are the key carriers delivering genetic information from one generation to the next. In a majority of animals, GCs segregate from somatic cells during embryogenesis by forming germlines. In land plants, GCs segregate from somatic cells during postembryonic development. In a majority of angiosperms, male GCs (archesporial cells) initiate at the four corners of the anther primordia. Little is known about the mechanism underlying this initiation. Here, we discovered that the dynamic auxin distribution in developing anthers coincided with GC initiation. A centripetal auxin gradient gradually formed toward the four corners where GCs will initiate. Local auxin biosynthesis was necessary for this patterning and for GC specification. The GC determinant protein SPOROCYTELESS/NOZZLE (SPL/NZZ) mediated the effect of auxin on GC specification and modified auxin biosynthesis to maintain a centripetal auxin distribution. Our work reveals that auxin is a key factor guiding GC specification in Arabidopsis anthers. Moreover, we demonstrate that the GC segregation from somatic cells is not a simple switch on/off event but rather a complicated process that involves a dynamic feedback circuit among local auxin biosynthesis, transcription of SPL/NZZ, and a progressive GC specification. This finding sheds light on the mystery of how zygote-derived somatic cells diverge into GCs in plants.

Gain-of-function of the 1-aminocyclopropane-1-carboxylate synthase gene ACS1G induces female flower development in cucumber gynoecy.

Unisexual flowers provide a useful system for studying plant sex determination. In cucumber (Cucumis sativus L.), three major Mendelian loci control unisexual flower development, Female (F), androecious [a; 1-aminocyclopropane-1-carboxylate {ACC} synthase 11, acs11], and Monoecious (M; ACS2), referred to here as the Female, Androecious, Monoecious (FAM) model, in combination with two genes, gynoecious (g, the WIP family C2H2 zinc finger transcription factor gene WIP1) and the ethylene biosynthetic gene ACC oxidase 2 (ACO2). The F locus, conferring gynoecy and the potential for increasing fruit yield, is defined by a 30.2-kb tandem duplication containing three genes. However, the gene that determines the Female phenotype, and its mechanism, remains unknown. Here, we created a set of mutants and revealed that ACS1G is responsible for gynoecy conferred by the F locus. The duplication resulted in ACS1G acquiring a new promoter and expression pattern; in plants carrying the F locus duplication, ACS1G is expressed early in floral bud development, where it functions with ACO2 to generate an ethylene burst. The resulting ethylene represses WIP1 and activates ACS2 to initiate gynoecy. This early ACS1G expression bypasses the need for ACS11 to produce ethylene, thereby establishing a dominant pathway for female floral development. Based on these findings, we propose a model for how these ethylene biosynthesis genes cooperate to control unisexual flower development in cucumber.

Key innovations in transition from homospory to heterospory.

Heterospory (i.e. dimorphic spores) is a long-lasting topic discussed in plant biology. It is observed in many of ferns, fern allies, and seed plants. The rise of heterospory and the mechanisms underlying its success in plant evolution are not clearly elucidated. In this short communication, an attempt is made to shed some light on these two questions.

Plant Morphogenesis 123: a renaissance in modern botany?

Plants are a group of multicellular organisms crucial for the biosphere on the Earth. In the 17th century, the founding fathers of modern botany viewed the bud as the basic unit undergoing the plant life cycle. However, for many understandable reasons, the dominant conceptual framework evolved away from the "bud-centered" viewpoint to a "plant-centered" viewpoint that treated the whole plant, consisting of numerous buds, as a unit and considered the entire plant to be the functional equivalent of an animal individual. While this "plant-centered" viewpoint is convenient and great progress has been made using this conceptual framework, some fundamental problems remain logically unsolvable. Previously, I have proposed a new conceptual framework for interpretation of plant morphogenesis, called Plant Morphogenesis 123, which revives a "bud-centered" viewpoint. The perspective of Plant Morphogenesis 123 allows us to address new questions regarding to the mechanisms of plant morphogenesis that are important, and technically accessible, but previously neglected under the "plant-centered" conceptual framework. In addition to describing these questions, I address a more fundamental question for further discussion: why do people study plants?

Histone Deacetylase HDA19 Affects Root Cortical Cell Fate by Interacting with SCARECROW.

The Arabidopsis (Arabidopsis thaliana) root epidermis is a simple model for investigating cell fate specification and pattern formation. In addition to regulatory networks consisting of transcription factors, histone deacetylases are also involved in the formation of cellular patterns. Here, we report thatHistone Deacetylase19 (HDA19) affects the root epidermal cellular pattern through regulation of cortical cell fate by interacting with SCARECROW (SCR). HDA19 binds to the DNA sequence upstream of SCR, as well as to those of several of SCR's target genes, and regulates their expression. Mutant lines of several SCR target genes show impaired patterns of epidermal differentiation and cortical cell division, similar to that of hda19 This work presents HDA19 and SCR as two further players in the regulation of cortical and epidermal cell specification and describes an additional function for SCR.

Characterization of the Ubiquitin C-Terminal Hydrolase and Ubiquitin-Specific Protease Families in Rice (Oryza sativa).

The ubiquitin C-terminal hydrolase (UCH) and ubiquitin-specific processing protease (UBP) protein families both function in protein deubiquitination, playing important roles in a wide range of biological processes in animals, fungi, and plants. Little is known about the functions of these proteins in rice (Oryza sativa), and the numbers of genes reported for these families have not been consistent between different rice database resources. To further explore their functions, it is necessary to first clarify the basic molecular and biochemical nature of these two gene families. Using a database similarity search, we clarified the numbers of genes in these two families in the rice genome, examined the enzyme activities of their corresponding proteins, and characterized the expression patterns of all OsUCH and representative OsUBP genes. Five OsUCH and 44 OsUBP genes were identified in the rice genome, with four OsUCH proteins and 10 of 16 tested representative OsUBP proteins showing enzymatic activities. Two OsUCHs and five OsUBPs were found to be preferentially expressed in the early development of rice stamens. This work thus lays down a reliable bioinformatic foundation for future investigations of genes in these two families, particularly for exploring their potential roles in rice stamen development.

Structure for energy cycle: a unique status of the second law of thermodynamics for living systems.

Distinguishing things from beings, or matters from lives, is a fundamental question. Extending E. Schrödinger's neg-entropy and I. Prigogine's dissipative structure, we propose a chemical kinetic view that the earliest "live" process is embedded essentially in a special interaction between a pair of specific components under a particular, corresponding environmental conditions. The interaction exists as an inter-molecular-force-bond complex (IMFBC) that couples two separate chemical processes: one is the spontaneous formation of the IMFBC driven by a decrease of Gibbs free energy as a dissipative process; while the other is the disassembly of the IMFBC driven thermodynamically by free energy input from the environment. The two chemical processes coupled by the IMFBC originated independently and were considered non-living on Earth, but the IMFBC coupling of the two can be considered as the earliest form of metabolism: the first landmark on the path from things to a being. The dynamic formation and disassembly of the IMFBC, as a composite individual, follows a principle designated as "… structure for energy for structure for energy…", the cycle continues; and for short it will be referred to as "structure for energy cycle". With additional features derived from this starting point, the IMFBC-centered "live" process spontaneously evolved into more complex living organisms with the characteristics currently known.

Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development.

This study aimed at elucidating regulatory components behind floral organ identity determination and tissue development. It remains unclear how organ identity proteins facilitate development of organ primordia into tissues with a determined identity, even though it has long been accepted that floral organ identity is genetically determined by interaction of identity genes according to the ABC model. Using the chromatin immunoprecipitation sequencing technique, we identified OsTGA10, encoding a bZIP transcription factor, as a target of the MADS box protein OsMADS8, which is annotated as an E-class organ identity protein. We characterized the function of OsTGA10 using genetic and molecular analyses. OsTGA10 was preferentially expressed during stamen development, and mutation of OsTGA10 resulted in male sterility. OsTGA10 was required for tapetum development and functioned by interacting with known tapetum genes. In addition, in ostga10 stamens, the hallmark cell wall thickening of the endothecium was defective. Our findings suggest that OsTGA10 plays a mediator role between organ identity determination and tapetum development in rice stamen development, between tapetum development and microspore development, and between various regulatory components required for tapetum development. Furthermore, the defective endothecium in ostga10 implies that cell wall thickening of endothecium is dependent on tapetum development.

Two types of germ cells, the sexual reproduction cycle, and the double-ring mode of plant developmental program.

In this viewpoint, the usages of terms for progenitor cells to meiosis and gametogenesis are discussed. Terms for 2 types of germ cells, i.e. "diploid germ cells" and "haploid germ cells" were suggested to replace "archesporial cells" and "generative cells," respectively, in plant developmental research. This suggestion was based on 2 newly proposed concepts, the "sexual reproduction cycle" for eukaryotes, and a "double-ring mode" of plant developmental program.

Expression Analyses of Embryogenesis-Associated Genes during Somatic Embryogenesis of Adiantum capillus-veneris L. In vitro: New Insights into the Evolution of Reproductive Organs in Land Plants.

An efficient in vitro regeneration system via somatic embryogenesis (SE) was developed for a fern species Adiantum capillus-veneris. Adventitious shoots, green globular bodies (GGBs) and calli were obtained with the maximal induction rate on the Murashige and Skoog (MS) medium of low concentrations of 6-benzyladenine (BA) (0-1.0 mg/L), 2.0 mg/L BA without 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/L 2,4-D and 0.5-1.0 mg/L 6-BA, respectively. Cyto-morphological and histological changes in the shoot development via calli and GGBs were examined. For a better understanding of these developmental events, expression patterns of six genes, AcLBD16, AcAGL, AcBBM, AcWUS, AcRKD, and AcLEC1, were characterized during SE. AcBBM and AcLEC1 were ubiquitously expressed in direct SE (adventitious shoots and GGBs) the maximal expression of AcBBM in mature GGBs, and the high expression of AcLEC1 in GGB initiation and adventitious shoots. During the indirect SE, AcLBD16, AcLEC1, AcRKD, and AcWUS were highly expressed in mature calli. Additionally, phylogenetic analyses showed that AcWUS, AcBBM, AcLBD, AcAGL, AcRKD, and their homologs of other green plants formed monophyletic clades, respectively. Some of these gene families, however, diversified rapidly with the occurrence of embryophytes, suggesting that embryogenesis-associated genes could experience a rapid evolution with the colonization of plants to terrestrial environments. Expression and phylogenetic analyses of those embryogenesis-associated genes by the aid of in vitro regeneration system of A. capillus-veneris provide new insights into the evolution of reproductive organs in land plants.

Sugar Treatments Can Induce AcLEAFY COTYLEDON1 Expression and Trigger the Accumulation of Storage Products during Prothallus Development of Adiantum capillus-veneris.

A seed is an intricate structure. Of the two development processes involved in seed formation, seed maturation, or seed program includes accumulation of storage products, acquisition of desiccation tolerance, and induction of dormancy. Little is known about how these processes were originated and integrated into the life cycle of seed plants. While previous investigation on seed origin was almost exclusively through fossil comparison in paleobotany, a wealth of information about the key role of LEAFY COTYLEDON1 (LEC1) in seed formation of spermatophyte inspired a new approach to investigating the seed origin mystery. Here, we examined the expression pattern of AcLEC1 during the entire life cycle of Adiantum capillus-veneris, a non-seed plant, confirmed no AcLEC1 gene expression detectable in prothalli, demonstrated inductive expressed by both sucrose and glucose in prothalli. As expected, we found that sugar treatments delayed prothallus development, promoted differentiation of reproductive organs, and triggered accumulation of storage products. These findings demonstrated links between the sugar treatments and the induction of AcLEC1 expression, as well as the sugar treatments and the events such as accumulation of storage products, which is similar to those considered as seed maturation process in seed plants. These links support a modified hypothesis that inductive expression of LEC1 homologs during embryogenesis might be a key innovation for the origin of the seed program.

Reconsideration of Plant Morphological Traits: From a Structure-Based Perspective to a Function-Based Evolutionary Perspective.

This opinion article proposes a novel alignment of traits in plant morphogenesis from a function-based evolutionary perspective. As a member species of the ecosystem on Earth, we human beings view our neighbor organisms from our own sensing system. We tend to distinguish forms and structures (i.e., "morphological traits") mainly through vision. Traditionally, a plant was considered to be consisted of three parts, i.e., the shoot, the leaves, and the root. Based on such a "structure-based perspective," evolutionary analyses or comparisons across species were made on particular parts or their derived structures. So far no conceptual framework has been established to incorporate the morphological traits of all three land plant phyta, i.e., bryophyta, pteridophyta and spermatophyta, for evolutionary developmental analysis. Using the tenets of the recently proposed concept of sexual reproduction cycle, the major morphological traits of land plants can be aligned into five categories from a function-based evolutionary perspective. From this perspective, and the resulting alignment, a new conceptual framework emerges, called "Plant Morphogenesis 123." This framework views a plant as a colony of integrated plant developmental units that are each produced via one life cycle. This view provided an alternative perspective for evolutionary developmental investigation in plants.