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.

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.

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.

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.

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.

Phosphorylation of SPOROCYTELESS/NOZZLE by the MPK3/6 Kinase Is Required for Anther Development.

Germ cells are indispensable carriers of genetic information from one generation to the next. In contrast to the well-understood process in animals, information on the mechanism of germ cell initiation in plants is very limited. SPOROCYTELESS/NOZZLE was previously identified as an essential regulator of diploid germ cell (archesporial cell) differentiation in the stamens and ovules of Arabidopsis (Arabidopsis thaliana). Although SPOROCYTELESS (SPL) transcription is activated by the floral organ identity regulator AGAMOUS and epigenetically regulated by SET DOMAIN GROUP2, little is known about the regulation of the SPL protein. Here, we report that the protein kinases MPK3 and MPK6 can both interact with SPL in vitro and in vivo and can phosphorylate the SPL protein in vitro. In addition, phosphorylation of the SPL protein by MPK3/6 is required for SPL function in the Arabidopsis anther, as measured by its effect on archesporial cell differentiation. We further demonstrate that phosphorylation enhances SPL protein stability. This work not only uncovers the importance of SPL phosphorylation for its regulatory role in Arabidopsis anther development, but also supports the hypothesis that the regulation of precise spatiotemporal patterning of germ cell initiation and that differentiation is achieved progressively through multiple levels of regulation, including transcriptional and posttranslational modification.

HISTONE DEACETYLASE6-Defective Mutants Show Increased Expression and Acetylation of ENHANCER OF TRIPTYCHON AND CAPRICE1 and GLABRA2 with Small But Significant Effects on Root Epidermis Cellular Pattern.

Cellular patterning in the Arabidopsis (Arabidopsis thaliana) root epidermis is dependent on positional information, the transmission of which involves histone acetylation. Here, we report that HISTONE DEACETYLASE6 (HDA6) has significant effects on this cellular patterning. Mutation of HDA6 led to ectopic hair cells in the nonhair positions of root epidermis in Arabidopsis, based on an analysis of paraffin sections stained with Toluidine Blue. While HDA6 was present throughout the root tip, epidermis-specific complementation with HDA6 could rescue the hda6 phenotype. Both transcript levels and expression patterns of ENHANCER OF TRIPTYCHON AND CAPRICE1 (ETC1) and GLABRA2 (GL2) in the root tip were affected in hda6. Consistent with these changes in expression, HDA6 directly bound to the promoter regions of ETC1 and GL2, and acetylation of histone H3 on these promoter regions and acetylation of histone H4 on the ETC1 promoter region was increased in the hda6 mutant. Taken together, these results indicate that HDA6 affects the cellular patterning of Arabidopsis root epidermis through altering the histone acetylation status of ETC1 and GL2 promoters and thereby affects the expression of these two components of the core transcription factor network determining epidermal cell fates. Our findings thus provide new insights into the role of histone acetylation in root epidermis cell patterning.

HDA18 affects cell fate in Arabidopsis root epidermis via histone acetylation at four kinase genes.

The differentiation of hair (H) and non-hair (N) cells in the Arabidopsis thaliana root epidermis is dependent on positional relationships with underlying cortical cells. We previously found that histone acetylation relays positional information and that a mutant altered in the histone deacetylase gene family member HISTONE DEACETYLASE 18 (HDA18) exhibits altered H and N epidermal cell patterning. Here, we report that HDA18 has in vitro histone deacetylase activity and that both mutation and overexpression of HDA18 led to cells at the N position having H fate. The HDA18 protein physically interacted with histones related to a specific group of kinase genes, which are demonstrated in this study to be components of a positional information relay system. Both down- and upregulation of HDA18 increased transcription of the targeted kinase genes. Interestingly, the acetylation levels of histone 3 lysine 9 (H3K9), histone 3 lysine 14 (H3K14) and histone 3 lysine 18 (H3K18) at the kinase genes were differentially affected by down- or upregulation of HDA18, which explains why the transcription levels of the four HDA18-target kinase genes increased in all lines with altered HDA18 expression. Our results reveal the surprisingly complex mechanism by which HDA18 affects cellular patterning in Arabidopsis root epidermis.

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.

Adjustment method for microarray data generated using two-cycle RNA labeling protocol.

BACKGROUND: Microarray technology is widely utilized for monitoring the expression changes of thousands of genes simultaneously. However, the requirement of relatively large amount of RNA for labeling and hybridization makes it difficult to perform microarray experiments with limited biological materials, thus leads to the development of many methods for preparing and amplifying mRNA. It is addressed that amplification methods usually bring bias, which may strongly hamper the following interpretation of the results. A big challenge is how to correct for the bias before further analysis. RESULTS: In this article, we observed the bias in rice gene expression microarray data generated with the Affymetrix one-cycle, two-cycle RNA labeling protocols, followed by validation with Real Time PCR. Based on these data, we proposed a statistical framework to model the processes of mRNA two-cycle linear amplification, and established a linear model for probe level correction. Maximum Likelihood Estimation (MLE) was applied to perform robust estimation of the Retaining Rate for each probe. After bias correction, some known pre-processing methods, such as PDNN, could be combined to finish preprocessing. Then, we evaluated our model and the results suggest that our model can effectively increase the quality of the microarray raw data: (i) Decrease the Coefficient of Variation for PM intensities of probe sets; (ii) Distinguish the microarray samples of five stages for rice stamen development more clearly; (iii) Improve the correlation coefficients among stamen microarray samples. We also discussed the necessity of model adjustment by comparing with another simple adjustment method. CONCLUSION: We conclude that the adjustment model is necessary and could effectively increase the quality of estimation for gene expression from the microarray raw data.

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.

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.

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.

Ethylene perception is involved in female cucumber flower development.

It is well established that ethylene promotes female flower development in cucumber. However, little is known about how the gaseous hormone selectively affects female flowers, and what mechanism it uses. Previously, we found organ-specific DNA damage in the primordial anther of female cucumber flowers. This finding led to a hypothesis that ethylene might promote female flower development via the organ-specific induction of DNA damage in primordial anthers. In this study, we tested this hypothesis first by demonstrating ethylene induction of DNA damage via the ethylene signaling pathway using cucumber protoplasts. Then, using representative component genes of the ethylene signaling pathway as probes, we found that one of the ethylene receptors, CsETR1, was temporally and spatially downregulated in the stamens of stage-6 female cucumber flowers, especially along with the increase of the nodes. Furthermore, by constructing transgenic Arabidopsis plants with organ-specific expression of antisense CsETR1 under the control of an AP3 promoter to downregulate ETR1 expression in the stamens, we generated Arabidopsis 'female flowers', in which the abnormal stamens mimic those of female cucumber flowers. Our data suggest that ethylene perception is involved in the arrest of stamen development in female cucumber flowers through the induction of DNA damage. This opens up a novel perspective and approach to solve the half-century-long puzzle of how gaseous ethylene selectively promotes female flowers in the monoecious cucumber plant.

Characterization of an ethylene-inducible, calcium-dependent nuclease that is differentially expressed in cucumber flower development.

• Production of unisexual flowers is an important mechanism that promotes cross-pollination in angiosperms. We previously identified primordial anther-specific DNA damage and organ-specific ethylene perception responsible for the arrest of stamen development in female flowers, but little is known about how the two processes are linked. • To identify potential links between the two processes, we performed suppression subtractive hybridization (SSH) on cucumber (Cucumis sativus L.) stamens of male and female flowers at stage 6, with stamens at stage 5 of bisexual flowers as a control. • Among the differentially expressed genes, we identified an expressed sequence tag (EST) encoding a cucumber homolog to an Arabidopsis calcium-dependent nuclease (CAN), designated CsCaN. Full-length CsCaN cDNA and the respective genomic DNA sequence were cloned and characterized. The CsCaN protein exhibited calcium-dependent nuclease activity. CsCaN showed ubiquitous expression; however, increased gene expression was detected in the stamens of stage 6 female flowers compared with male flowers. As expected, CsCaN expression was ethylene inducible. It was of great interest that CsCaN was post-translationally modified. • This study demonstrated that CsCaN is a novel cucumber nuclease gene, whose DNase activity is regulated at multiple levels, and which could be involved in the primordial anther-specific DNA damage of developing female cucumber flowers.

Duplication and functional diversification of HAP3 genes leading to the origin of the seed-developmental regulatory gene, LEAFY COTYLEDON1 (LEC1), in nonseed plant genomes.

The HAP3 gene encodes a subunit of the CCAAT-box-binding factor (CBF), a highly conserved trimeric activator that recognizes and binds the ubiquitous CCAAT promoter element with high affinity. Two types of HAP3 gene have been identified in plant genomes. The LEAFY COTYLEDON1 (LEC1)-type HAP3 genes encode a functionally specialized subunit of CBF, which is expressed specifically in developing seeds. In contrast, most non-LEC1-type HAP3 genes are expressed in various tissues. It has been proposed that the LEC1-type HAP3 genes originated from the duplication and functional divergence of non-LEC1-type HAP3 genes. However, it is not yet known when this duplication event took place or whether the LEC1-type HAP3 genes appeared at the same time as the origin of seed plants. Here we describe a comprehensive comparison of the duplication patterns of HAP3 genes in different plant genomes. We recognize a major expansion of the HAP3 gene family accompanying the origin and early diversification of land plants and postulate that retrotransposition and other mechanisms of gene duplication have been involved in the expansion of the plant HAP3 gene family. We provide evidence that the LEC1-type HAP3 genes originated in nonseed vascular plant genomes and demonstrate that they are inductively expressed under drought stress in nonseed plants. These genes, however, were recruited to a novel regulatory network in the early stages of seed plant evolution and steadily expressed during seed development and maturation.

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?