Antagonistic regulation of the gibberellic acid response during stem growth in rice.

The size of plants is largely determined by growth of the stem. Stem elongation is stimulated by gibberellic acid1-3. Here we show that internode stem elongation in rice is regulated antagonistically by an 'accelerator' and a 'decelerator' in concert with gibberellic acid. Expression of a gene we name ACCELERATOR OF INTERNODE ELONGATION 1 (ACE1), which encodes a protein of unknown function, confers cells of the intercalary meristematic region with the competence for cell division, leading to internode elongation in the presence of gibberellic acid. By contrast, upregulation of DECELERATOR OF INTERNODE ELONGATION 1 (DEC1), which encodes a zinc-finger transcription factor, suppresses internode elongation, whereas downregulation of DEC1 allows internode elongation. We also show that the mechanism of internode elongation that is mediated by ACE1 and DEC1 is conserved in the Gramineae family. Furthermore, an analysis of genetic diversity suggests that mutations in ACE1 and DEC1 have historically contributed to the selection of shorter plants in domesticated populations of rice to increase their resistance to lodging, and of taller plants in wild species of rice for adaptation to growth in deep water. Our identification of these antagonistic regulatory factors enhances our understanding of the gibberellic acid response as an additional mechanism that regulates internode elongation and environmental fitness, beyond biosynthesis and gibberellic acid signal transduction.

Genome-wide cis-decoding for expression design in tomato using cistrome data and explainable deep learning.

In the evolutionary history of plants, variation in cis-regulatory elements (CREs) resulting in diversification of gene expression has played a central role in driving the evolution of lineage-specific traits. However, it is difficult to predict expression behaviors from CRE patterns to properly harness them, mainly because the biological processes are complex. In this study, we used cistrome datasets and explainable convolutional neural network (CNN) frameworks to predict genome-wide expression patterns in tomato (Solanum lycopersicum) fruit from the DNA sequences in gene regulatory regions. By fixing the effects of trans-acting factors using single cell-type spatiotemporal transcriptome data for the response variables, we developed a prediction model for crucial expression patterns in the initiation of tomato fruit ripening. Feature visualization of the CNNs identified nucleotide residues critical to the objective expression pattern in each gene, and their effects were validated experimentally in ripening tomato fruit. This cis-decoding framework will not only contribute to the understanding of the regulatory networks derived from CREs and transcription factor interactions, but also provides a flexible means of designing alleles for optimized expression.

A leaf-emanated signal orchestrates grain size and number in response to maternal resources.

In plants, variations in seed size and number are outcomes of different reproductive strategies. Both traits are often environmentally influenced, suggesting that a mechanism exists to coordinate these phenotypes in response to available maternal resources. Yet, how maternal resources are sensed and influence seed size and number is largely unknown. Here, we report a mechanism that senses maternal resources and coordinates grain size and number in the wild rice Oryza rufipogon, a wild progenitor of Asian cultivated rice. We showed that FT-like 9 (FTL9) regulates both grain size and number and that maternal photosynthetic assimilates induce FTL9 expression in leaves to act as a long-range signal that increases grain number and reduces size. Our findings highlight a strategy that benefits wild plants to survive in a fluctuating environment. In this strategy, when maternal resources are sufficient, wild plants increase their offspring number while preventing an increase in offspring size by the action of FTL9, which helps expand their habitats. In addition, we found that a loss-of-function allele (ftl9) is prevalent among wild and cultivated populations, offering a new scenario in the history of rice domestication.

Ongoing Rapid Evolution of a Post-Y Region Revealed by Chromosome-Scale Genome Assembly of a Hexaploid Monoecious Persimmon (Diospyros kaki).

Plants have evolved sex chromosomes independently in many lineages, and loss of separate sexes can also occur. In this study, we assembled a monoecious recently hexaploidized persimmon (Diospyros kaki), in which the Y chromosome has lost the maleness-determining function. Comparative genomic analysis of D. kaki and its dioecious relatives uncovered the evolutionary process by which the nonfunctional Y chromosome (or Ymonoecy) was derived, which involved silencing of the sex-determining gene, OGI, approximately 2 million years ago. Analyses of the entire X and Ymonoecy chromosomes suggested that D. kaki's nonfunctional male-specific region of the Y chromosome (MSY), which we call a post-MSY, has conserved some characteristics of the original functional MSY. Specifically, comparing the functional MSY in Diospyros lotus and the nonfunctional "post-MSY" in D. kaki indicated that both have been rapidly rearranged, mainly via ongoing transposable element bursts, resembling structural changes often detected in Y-linked regions, some of which can enlarge the nonrecombining regions. The recent evolution of the post-MSY (and possibly also MSYs in dioecious Diospyros species) therefore probably reflects these regions' ancestral location in a pericentromeric region, rather than the presence of male-determining genes and/or genes controlling sexually dimorphic traits.

Transcriptomic Interpretation on Explainable AI-Guided Intuition Uncovers Premonitory Reactions of Disordering Fate in Persimmon Fruit.

Deep neural network (DNN) techniques, as an advanced machine learning framework, have allowed various image diagnoses in plants, which often achieve better prediction performance than human experts in each specific field. Notwithstanding, in plant biology, the application of DNNs is still mostly limited to rapid and effective phenotyping. The recent development of explainable CNN frameworks has allowed visualization of the features in the prediction by a convolutional neural network (CNN), which potentially contributes to the understanding of physiological mechanisms in objective phenotypes. In this study, we propose an integration of explainable CNN and transcriptomic approach to make a physiological interpretation of a fruit internal disorder in persimmon, rapid over-softening. We constructed CNN models to accurately predict the fate to be rapid softening in persimmon cv. Soshu, only with photo images. The explainable CNNs, such as Gradient-weighted Class Activation Mapping (Grad-Class Activation Mapping (CAM)) and guided Grad-CAM, visualized specific featured regions relevant to the prediction of rapid softening, which would correspond to the premonitory symptoms in a fruit. Transcriptomic analyses to compare the featured regions of the predicted rapid-softening and control fruits suggested that rapid softening is triggered by precocious ethylene signal-dependent cell wall modification, despite exhibiting no direct phenotypic changes. Further transcriptomic comparison between the featured and non-featured regions in the predicted rapid-softening fruit suggested that premonitory symptoms reflected hypoxia and the related stress signals finally to induce ethylene signals. These results would provide a good example for the collaboration of image analysis and omics approaches in plant physiology, which uncovered a novel aspect of fruit premonitory reactions in the rapid-softening fate.

The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants.

Most angiosperms bear hermaphroditic flowers, but a few species have evolved outcrossing strategies, such as dioecy, the presence of separate male and female individuals. We previously investigated the mechanisms underlying dioecy in diploid persimmon (D. lotus) and found that male flowers are specified by repression of the autosomal gene MeGI by its paralog, the Y-encoded pseudo-gene OGI. This mechanism is thought to be lineage-specific, but its evolutionary path remains unknown. Here, we developed a full draft of the diploid persimmon genome (D. lotus), which revealed a lineage-specific whole-genome duplication event and provided information on the architecture of the Y chromosome. We also identified three paralogs, MeGI, OGI and newly identified Sister of MeGI (SiMeGI). Evolutionary analysis suggested that MeGI underwent adaptive evolution after the whole-genome duplication event. Transformation of tobacco plants with MeGI and SiMeGI revealed that MeGI specifically acquired a new function as a repressor of male organ development, while SiMeGI presumably maintained the original function. Later, a segmental duplication event spawned MeGI's regulator OGI on the Y-chromosome, completing the path leading to dioecy, and probably initiating the formation of the Y-chromosome. These findings exemplify how duplication events can provide flexible genetic material available to help respond to varying environments and provide interesting parallels for our understanding of the mechanisms underlying the transition into dieocy in plants.

Correction: The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants.

[This corrects the article DOI: 10.1371/journal.pgen.1008566.].

Evolution of sex in crops: recurrent scrap and rebuild.

Sexuality is the main strategy for maintaining genetic diversity within a species. In flowering plants (angiosperms), sexuality is derived from ancestral hermaphroditism and multiple sexualities can be expressed in an individual. The mechanisms conferring chromosomal sex determination in plants (or dioecy) have been studied for over a century by both biologists and agricultural scientists, given the importance of this field for crop cultivation and breeding. Despite extensive research, the sex determining gene(s) in plants had not been identified until recently. In this review, we dissect plant sex evolution and determining systems, with a focus on crop species. We introduced classic studies with theoretical, genetic, and cytogenic approaches, as well as more recent research using advanced molecular and genomic techniques. Plants have undergone very frequent transitions into, and out of, dioecy. Although only a few sex determinants have been identified in plants, an integrative viewpoint on their evolutionary trends suggests that recurrent neofunctionalization events are potentially common, in a "scrap and (re)build" cycle. We also discuss the potential association between crop domestication and transitions in sexual systems. We focus on the contribution of duplication events, which are particularly frequent in plant taxa, as a trigger for the creation of new sexual systems.

Validation of pencil beam scanning proton therapy with multi-leaf collimator calculated by a commercial Monte Carlo dose engine.

This study aimed to evaluate the clinical beam commissioning results and lateral penumbra characteristics of our new pencil beam scanning (PBS) proton therapy using a multi-leaf collimator (MLC) calculated by use of a commercial Monte Carlo dose engine. Eighteen collimated uniform dose plans for cubic targets were optimized by the RayStation 9A treatment planning system (TPS), varying scan area, modulation widths, measurement depths, and collimator angles. To test the patient-specific measurements, we also created and verified five clinically realistic PBS plans with the MLC, such as the liver, prostate, base-of-skull, C-shape, and head-and-neck. The verification measurements consist of the depth dose (DD), lateral profile (LP), and absolute dose (AD). We compared the LPs and ADs between the calculation and measurements. For the cubic plans, the gamma index pass rates (γ-passing) were on average 96.5% ± 4.0% at 3%/3 mm for the DD and 95.2% ± 7.6% at 2%/2 mm for the LP. In several LP measurements less than 75 mm depths, the γ-passing deteriorated (increased the measured doses) by less than 90% with the scattering such as the MLC edge and range shifter. The deteriorated γ-passing was satisfied by more than 90% at 2%/2 mm using uncollimated beams instead of collimated beams except for three planes. The AD differences and the lateral penumbra width (80%-20% distance) were within ±1.9% and ± 1.1 mm, respectively. For the clinical plan measurements, the γ-passing of LP at 2%/2 mm and the AD differences were 97.7% ± 4.2% on average and within ±1.8%, respectively. The measurements were in good agreement with the calculations of both the cubic and clinical plans inserted in the MLC except for LPs less than 75 mm regions of some cubic and clinical plans. The calculation errors in collimated beams can be mitigated by substituting uncollimated beams.

Interspecific introgression and natural selection in the evolution of Japanese apricot (Prunus mume).

Domestication and population differentiation in crops involve considerable phenotypic changes. The logs of these evolutionary paths, including natural/artificial selection, can be found in the genomes of the current populations. However, these profiles have been little studied in tree crops, which have specific characters, such as long generation time and clonal propagation, maintaining high levels of heterozygosity. We conducted exon-targeted resequencing of 129 genomes in the genus Prunus, mainly Japanese apricot (Prunus mume), and apricot (Prunus armeniaca), plum (Prunus salicina), and peach (Prunus persica). Based on their genome-wide single-nucleotide polymorphisms merged with published resequencing data of 79 Chinese P. mume cultivars, we inferred complete and ongoing population differentiation in P. mume. Sliding window characterization of the indexes for genetic differentiation identified interspecific fragment introgressions between P. mume and related species (plum and apricot). These regions often exhibited strong selective sweeps formed in the paths of establishment or formation of substructures of P. mume, suggesting that P. mume has frequently imported advantageous genes from other species in the subgenus Prunus as adaptive evolution. These findings shed light on the complicated nature of adaptive evolution in a tree crop that has undergone interspecific exchange of genome fragments with natural/artificial selections.

Shy Girl, a kiwifruit suppressor of feminization, restricts gynoecium development via regulation of cytokinin metabolism and signalling.

Kiwifruit (Actinidia chinensis) is a dioecious, long-living woody perennial vine. Reduced generation time and induction of hermaphroditism can accelerate crop improvement and facilitate alternative farming for better food security in the face of climate change. Previous studies identified that CENTRORADIALIS genes CEN and CEN4 act to repress flowering, whilst the male-specific Shy Girl (SyGl) gene with homology to type-C cytokinin response regulators could repress gynoecium development in model plants. Here we use CRISPR/Cas9 to mutagenize CEN, CEN4 and SyGl in the male kiwifruit A. chinensis 'Bruce'. Biallelic mutations of CEN and CEN4 generated rapid-flowering male plants, and simultaneous targeting of CEN4 and SyGl gave rise to rapid-flowering hermaphrodites with restored gynoecial function and viable pollen, providing functional evidence for the role of SyGl in suppression of feminization. Analysis of ovary tissues identified genes that contribute to carpel development and revealed that SyGl affected both cytokinin profiles and the expression of genes involved in cytokinin metabolism and signalling. The plant lines generated by CEN4/SyGl knockout could self-pollinate and produce fast-flowering offspring. These results establish that SyGI acts as the suppressor of feminization in kiwifruit and demonstrate the potential for accelerated breeding in an outcrossing horticultural woody perennial.

A Y-Encoded Suppressor of Feminization Arose via Lineage-Specific Duplication of a Cytokinin Response Regulator in Kiwifruit.

Dioecy, the presence of male and female flowers on distinct individuals, has evolved independently in multiple plant lineages, and the genes involved in this differential development are just starting to be uncovered in a few species. Here, we used genomic approaches to investigate this pathway in kiwifruits (genus Actinidia). Genome-wide cataloging of male-specific subsequences, combined with transcriptome analysis, led to the identification of a type-C cytokinin response regulator as a potential sex determinant gene in this genus. Functional transgenic analyses in two model systems, Arabidopsis thaliana and Nicotiana tabacum, indicated that this gene acts as a dominant suppressor of carpel development, prompting us to name it Shy Girl (SyGI). Evolutionary analyses in a panel of Actinidia species revealed that SyGI is located in the Y-specific region of the genome and probably arose from a lineage-specific gene duplication. Comparisons with the duplicated autosomal counterpart, and with orthologs from other angiosperms, suggest that the SyGI-specific duplication and subsequent evolution of cis-elements may have played a key role in the acquisition of separate sexes in this species.

Evolution and diversification of the plant gibberellin receptor GID1.

The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.

Increase in the intensity of an optical signal with fluorescein during proton and carbon-ion irradiation.

PURPOSE: Although the imaging of luminescence emitted in water during irradiation of protons and carbon ions is a useful method for range and dose estimations, the intensity of the images is relatively low due to the low photon production of the luminescence phenomenon. Therefore, a relatively long time is required for the imaging. Since a fluorescent dye, fluorescein, may increase the intensity of the optical signal, we measured the luminescence images of water with different concentrations of fluorescein during irradiation of protons and carbon ions and compared the results with those by measurements with water. METHODS: A cooled charge-coupled device (CCD) camera was used for imaging a water phantom with different concentrations of fluorescein from 0.0063 to 0.025 mg/cm3 , in addition to a water phantom without fluorescein during irradiation of 150-MeV protons and 241.5-MeV/n carbon ions. RESULTS: For both protons and carbon ions, the intensity of the luminescence images increased as the concentration of fluorescein increased. With a fluorescein concentration of 0.025 mg/cm3 , the intensities increased to more than 10 times those of water for both protons and carbon ions. Although the shape of the depth profiles of luminescence images of water with fluorescein appeared similar to that of water for protons, those for carbon ions were different from those of water due to the increase in the Cherenkov light component at shallow depths by the decrease in the angular dependencies of the Cherenkov light. CONCLUSION: We confirmed the increase in intensity of the luminescence of water by adding fluorescein for particle ions. With a small amount of Cherenkov light contamination in the images, such as protons, the relative distributions of the luminescence images with fluorescein were similar to that of water and will be used for range or dose determination in a short time.

Gene networks orchestrated by MeGI: a single-factor mechanism underlying sex determination in persimmon.

Separating male and female sex organs is one of the main strategies used to maintain genetic diversity within a species. However, the genetic determinants and their regulatory mechanisms have been identified in only a few species. In dioecious persimmons, the homeodomain transcription factor, MeGI, which is the target of a Y chromosome-encoded small-RNA, OGI, can determine floral sexuality. The basic features of this system are conserved in the monoecious hexaploid Oriental persimmon, in which an additional epigenetic regulation of MeGI determines floral sexuality. The downstream regulatory pathways of MeGI remain uncharacterized. In this study, we examined transcriptomic data for male and female flowers from monoecious persimmon cultivars to unveil the gene networks orchestrated by MeGI. A network visualization and cistrome assessment suggested that class-1 KNOTTED-like homeobox (KNOX)/ovate family protein (OFP)/growth regulating factors (GRFs) and short vegetative phase (SVP) genes mediate the differences in gynoecium and androecium development between male and female flowers, respectively. The expression of these genes is directly controlled by MeGI. The gene networks also suggested that some cytokinin, auxin, and gibberellin signaling genes function cooperatively in the KNOX/OFP/GRF pathway during gynoecium differentiation. Meanwhile, SVP may repress PI expression in developing androecia. Overall, our results suggest that MeGI evolved the ability to promote gynoecium development and suppress androecium development by regulating KNOX/OFP/GRF and SVP expression levels, respectively. These insights may help to clarify the molecular mechanism underlying the production of unisexual flowers, while also elucidating the physiological background enabling a single-factor system to establish dioecy in plants.

Epigenetic Flexibility Underlies Somaclonal Sex Conversions in Hexaploid Persimmon.

Epigenetic regulation adds a flexible layer to genetic variations, potentially enabling long-term, but reversible, changes to a trait, while maintaining genetic information. In the hexaploid Oriental persimmon (Diospyros kaki), genetically monoecious cultivars bearing male flowers require the Y-encoded small RNA (smRNA) gene, OGI. This gene represses the expression of its autosomal counterpart gene, MeGI, as part of the canonical male production system. However, a D. kaki cultivar, Saijo, which lacks the OGI gene and originally bears only female flowers, occasionally produces somaclonal mutant male and revertant female (RF) branches. In this study, we investigated the mechanisms underlying these somaclonal sex conversions in persimmon. Specifically, we aimed to unravel how a genetically female tree without the OGI gene can produce male flowers and RF flowers. Applying multi-omics approaches, we revealed that this noncanonical male production system is basically consistent with the canonical system, in which the accumulation of smRNA targeting MeGI and the considerable DNA methylation of MeGI are involved. The epigenetic status of MeGI on CGN and CHG was synchronized to the genome-wide methylation patterns, both in transition to and from the male production system. These results suggest that the somaclonal sex conversions in persimmon are driven by the genome-wide epigenetic regulatory activities. Moreover, flexibility in the epigenetic layers of long-lived plant species (e.g. trees) is important for overcoming genetic robustness.

Explainable Deep Learning Reproduces a 'Professional Eye' on the Diagnosis of Internal Disorders in Persimmon Fruit.

Recent rapid progress in deep neural network techniques has allowed recognition and classification of various objects, often exceeding the performance of the human eye. In plant biology and crop sciences, some deep neural network frameworks have been applied mainly for effective and rapid phenotyping. In this study, beyond simple optimizations of phenotyping, we propose an application of deep neural networks to make an image-based internal disorder diagnosis that is hard even for experts, and to visualize the reasons behind each diagnosis to provide biological interpretations. Here, we exemplified classification of calyx-end cracking in persimmon fruit by using five convolutional neural network models with various layer structures and examined potential analytical options involved in the diagnostic qualities. With 3,173 visible RGB images from the fruit apex side, the neural networks successfully made the binary classification of each degree of disorder, with up to 90% accuracy. Furthermore, feature visualizations, such as Grad-CAM and LRP, visualize the regions of the image that contribute to the diagnosis. They suggest that specific patterns of color unevenness, such as in the fruit peripheral area, can be indexes of calyx-end cracking. These results not only provided novel insights into indexes of fruit internal disorders but also proposed the potential applicability of deep neural networks in plant biology.

Low temperature modulates natural peel degreening in lemon fruit independently of endogenous ethylene.

Peel degreening is an important aspect of fruit ripening in many citrus fruit, and previous studies have shown that it can be advanced by ethylene treatment or by low-temperature storage. However, the important regulators and pathways involved in natural peel degreening remain largely unknown. To determine how natural peel degreening is regulated in lemon fruit (Citrus limon), we studied transcriptome and physiochemical changes in the flavedo in response to ethylene treatment and low temperatures. Treatment with ethylene induced rapid peel degreening, which was strongly inhibited by the ethylene antagonist, 1-methylcyclopropene (1-MCP). Compared with 25 ºC, moderately low storage temperatures of 5-20 °C also triggered peel degreening. Surprisingly, repeated 1-MCP treatments failed to inhibit the peel degreening induced by low temperature. Transcriptome analysis revealed that low temperature and ethylene independently regulated genes associated with chlorophyll degradation, carotenoid metabolism, photosystem proteins, phytohormone biosynthesis and signalling, and transcription factors. Peel degreening of fruit on trees occurred in association with drops in ambient temperature, and it coincided with the differential expression of low temperature-regulated genes. In contrast, genes that were uniquely regulated by ethylene showed no significant expression changes during on-tree peel degreening. Based on these findings, we hypothesize that low temperature plays a prominent role in regulating natural peel degreening independently of ethylene in citrus fruit.

Evolution of Lineage-Specific Gene Networks Underlying the Considerable Fruit Shape Diversity in Persimmon.

The shapes of plant organs reflect the evolution of each lineage and have been diversified according to lineage-specific adaptations to environment. Research on the molecular pathways responsible for organ shapes has traditionally been focused mainly on leaves or flowers. Thus, little is known about the pathways controlling fruit shapes, despite their diversity in some plant species. In this study, we analyzed oriental persimmon (Diospyros kaki), which exhibits considerable diversity in fruit shapes among cultivars, to elucidate the underlying molecular mechanism using transcriptomic data and quantitative evaluation. First, to filter the candidate genes associated with persimmon fruit shapes, the whole gene expression patterns obtained using mRNA-Seq analysis from 100 individuals, including a segregated population and various cultivars, were assessed to detect correlations with principal component scores for fruit shapes characterized with elliptic Fourier descriptors. Next, a gene co-expression network analysis with weighted gene co-expression network analysis (WGCNA) package revealed that class 1 KNOX family genes and SEEDSTICK function as integrators along with some phytohormone-related genes, to regulate the fruit shape diversity. On the other hand, the OVATE family genes also contribute to fruit shape diversity, of which pathway would be potentially shared with other plant species. Evolutionary aspects suggest that acquisition of a high lineage-specific and variable expression of class 1 KNOX gene, knotted-like homeobox of Arabidopsis thaliana 1 (KNAT1), in young fruit is important for establishing the persimmon-specific mechanism that determines fruit shape diversity.

Pleiotropic effects of sex-determining genes in the evolution of dioecy in two plant species.

One reason for studying sex chromosomes of flowering plants is that they have often evolved separate sexes recently, and the genomes of dioecious species may not yet have evolved adaptations to their changes from the ancestral state. An unstudied question concerns the relative importance of such adaptation, versus the effects of the mutations that led to separate sexes in the first place. Theoretical models for such an evolutionary change make the prediction that the mutations that created males must have sexually antagonistic effects, not only abolishing female functions, but also increasing male functions relative to the ancestral functional hermaphrodites. It is important to test this critical assumption. Moreover, the involvement of sexual antagonism also implies that plant sex-determining genes may directly cause some of the sexual dimorphisms observed in dioecious plants. Sex-determining genes are starting to be uncovered in plants, including species in the genera Diospyros and Actinidia (families Ebenaceae and Actinidiaceae, respectively). Here, we describe transgenic experiments in which the effects of the very different male-determining genes of these two dioecious species were studied in a non-dioecious plant, Nicotiana tabacum. The results indeed support the critical assumption outlined above.