Mind the (CRISPR) gaps: The European Commission's proposal for the use of NGTs in the EU.

The proposal by the European Commission to regulate New Genome Technique (NGT) plants is a leap forward, but it does not revise the current legislation on GMOs and includes many inconsistencies that may hinder the adoption of specific NGTs.

Polyploidy Affects Plant Growth and Alters Cell Wall Composition.

Polyploidization has played a key role in plant breeding and crop improvement. Although its potential to increase biomass yield is well described, the effect of polyploidization on biomass composition has largely remained unexplored. Here, we generated a series of Arabidopsis (Arabidopsis thaliana) plants with different somatic ploidy levels (2n, 4n, 6n, and 8n) and performed rigorous phenotypic characterization. Kinematic analysis showed that polyploids developed slower compared to diploids; however, tetra- and hexaploids, but not octaploids, generated larger rosettes due to delayed flowering. In addition, morphometric analysis of leaves showed that polyploidy affected epidermal pavement cells, with increased cell size and reduced cell number per leaf blade with incrementing ploidy. However, the inflorescence stem dry weight was highest in tetraploids. Cell wall characterization revealed that the basic somatic ploidy level negatively correlated with lignin and cellulose content, and positively correlated with matrix polysaccharide content (i.e. hemicellulose and pectin) in the stem tissue. In addition, higher ploidy plants displayed altered sugar composition. Such effects were linked to the delayed development of polyploids. Moreover, the changes in polyploid cell wall composition promoted saccharification yield. The results of this study indicate that induction of polyploidy is a promising breeding strategy to further tailor crops for biomass production.

Sweet Immunity: The Effect of Exogenous Fructans on the Susceptibility of Apple (Malus × domestica Borkh.) to Venturia inaequalis.

There is an urgent need for novel, efficient and environmentally friendly strategies to control apple scab (Venturia inaequalis), for the purpose of reducing overall pesticide use. Fructans are recently emerging as promising "priming" compounds, standing out for their safety and low production costs. The objective of this work was to test a fructan-triggered defense in the leaves of apple seedlings. It was demonstrated that exogenous leaf spraying can reduce the development of apple scab disease symptoms. When evaluated macroscopically and by V. inaequalis-specific qPCR, levan-treated leaves showed a significant reduction of sporulation and V. inaequalis DNA in comparison to mock- and inulin-treated leaves, comparable to the levels in fosetyl-aluminum-treated leaves. Furthermore, we observed a significant reduction of in vitro mycelial growth of V. inaequalis on plates supplemented with levans when compared to controls, indicating a direct inhibition of fungal growth. Variations in endogenous sugar contents in the leaves were followed during priming and subsequent infection, revealing complex dynamics as a function of time and leaf ontogeny. Our data are discussed in view of the present theories on sugar signaling and fructan-based immunity, identifying areas for future research and highlighting the potential use of fructans in apple scab management in orchards.

Cold Influences Male Reproductive Development in Plants: A Hazard to Fertility, but a Window for Evolution.

Being sessile organisms, plants suffer from various abiotic stresses including low temperature. In particular, male reproductive development of plants is extremely sensitive to cold which may dramatically reduce viable pollen shed and plant fertility. Cold stress disrupts stamen development and prominently interferes with the tapetum, with the stress-responsive hormones ABA and gibberellic acid being greatly involved. In particular, low temperature stress delays and/or inhibits programmed cell death of the tapetal cells which consequently damages pollen development and causes male sterility. On the other hand, studies in Arabidopsis and crops have revealed that ectopically decreased temperature has an impact on recombination and cytokinesis during meiotic cell division, implying a putative role for temperature in manipulating plant genomic diversity and architecture during the evolution of plants. Here, we review the current understanding of the physiological impact of cold stress on the main male reproductive development processes including tapetum development, male meiosis and gametogenesis. Moreover, we provide insights into the genetic factors and signaling pathways that are involved, with putative mechanisms being discussed.

PROTEIN PHOSHATASE 2A B'α and ß Maintain Centromeric Sister Chromatid Cohesion during Meiosis in Arabidopsis.

The correct separation of homologous chromosomes during meiosis I, and sister chromatids during meiosis II, relies on the tight control of the cohesion complex. The phosphorylation and subsequent cleavage of the meiotic recombination protein REC8 (REC8-like family protein [SYN1] in Arabidopsis [Arabidopsis thaliana]), the α-kleisin subunit of the cohesion ring, along the chromosome arms at meiosis I allows crossovers and separation of homologous chromosomes without chromatid dissociation. REC8 continues to localize and function at the centromeres up to metaphase II and, in yeast and vertebrates, is protected from cleavage by means of protein phosphatase 2A (PP2A)-mediated dephosphorylation. Here, we show that, in plants, centromeric sister chromatid cohesion until meiosis II also requires the activity of a PP2A-type phosphatase complex. The combined absence of the regulatory subunits PP2AB'α and PP2AB'ß leads to the premature loss of chromosome cohesion in meiosis I. Male meiocytes of the pp2ab'αß double mutant display premature depletion of SYN1. The PP2AA1 structural and B'α regulatory subunit localize specifically to centromeres until metaphase II, supporting a role for the PP2A complex in the SYN1-mediated maintenance of centromeric cohesion in plant meiosis.

Gibberellin Induces Diploid Pollen Formation by Interfering with Meiotic Cytokinesis.

The plant hormone gibberellic acid (GA) controls many physiological processes, including cell differentiation, cell elongation, seed germination, and response to abiotic stress. In this study, we report that exogenous treatment of flowering Arabidopsis (Arabidopsis thaliana) plants with GA specifically affects the process of male meiotic cytokinesis leading to meiotic restitution and the production of diploid (2n) pollen grains. Similar defects in meiotic cell division and reproductive ploidy stability occur in Arabidopsis plants depleted of RGA and GAI, two members of the DELLA family that function as suppressor of GA signaling. Cytological analysis of the double rga-24 gai-t6 mutant revealed that defects in male meiotic cytokinesis are not caused by alterations in meiosis I (MI or meiosis II (MII) chromosome dynamics, but instead result from aberrations in the spatial organization of the phragmoplast-like radial microtubule arrays (RMAs) at the end of meiosis II. In line with a role for GA in the genetic regulation of the male reproductive system, we additionally show that DELLA downstream targets MYB33 and MYB65 are redundantly required for functional RMA biosynthesis and male meiotic cytokinesis. By analyzing the expression of pRGA::GFP-RGA in the wild-type Landsberg erecta background, we demonstrate that the GFP-RGA protein is specifically expressed in the anther cell layers surrounding the meiocytes and microspores, suggesting that appropriate GA signaling in the somatic anther tissue is critical for male meiotic cell wall formation and thus plays an important role in consolidating the male gametophytic ploidy consistency.

Cold interferes with male meiotic cytokinesis in Arabidopsis thaliana independently of the AHK2/3-AHP2/3/5 cytokinin signaling module.

Previously we have shown that low temperature stress in Arabidopsis causes defects in microtubule organization and cytokinesis in male meiocytes, which leads to the formation of diploid pollen. Because cytokinin (CK) mediates multiple physiological responses to cold stress, we investigated whether CK signaling is involved in cold-induced diploid pollen formation. To this end, we monitored male sporogenesis in a series of mutants defective in CK metabolism and signalling. Arabidopsis plants with altered CK homeostasis, that is, the ahk2-2 ahk3-3 double and the ahp2-1 ahp3 ahp5-2 triple mutant, were cold sensitive and displayed similar defective male meiotic cytokinesis as wild type plants upon cold stress. These findings demonstrate that the AHK2/3-AHP2/3/5 CK-signaling module is not required for cold-induced ploidy stability of male gamete in Arabidopsis. Cytological analysis further revealed that the cold-induced cytokinesis defects in the ahk2-2 ahk3-3 mutant correlated with irregular organization of the radial microtubule array (RMA) in tetrad microspores at the end of male meiosis. Contrary to the ahk and ahp mutants, Arabidopsis plants defective for ARR1, a downstream target of ahk and ahp mediated CK signalling, displayed higher cold-tolerance of male meiotic cytokinesis program. We here suggest that the transcription regulator ARR1 may act independently from the CK AHK2/3-AHP2/3/5 signaling module in conveying the cold response to male meiocytes.

CENH3-GFP: a visual marker for gametophytic and somatic ploidy determination in Arabidopsis thaliana.

BACKGROUND: The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell's chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo. RESULTS: By modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events. CONCLUSIONS: This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.

GLUCAN SYNTHASE-LIKE8 and STEROL METHYLTRANSFERASE2 are required for ploidy consistency of the sexual reproduction system in Arabidopsis.

In sexually reproducing plants, the meiocyte-producing archesporal cell lineage is maintained at the diploid state to consolidate the formation of haploid gametes. In search of molecular factors that regulate this ploidy consistency, we isolated an Arabidopsis thaliana mutant, called enlarged tetrad2 (et2), which produces tetraploid meiocytes through the stochastic occurrence of premeiotic endomitosis. Endomitotic polyploidization events were induced by alterations in cell wall formation, and similar cytokinetic defects were sporadically observed in other tissues, including cotyledons and leaves. ET2 encodes GLUCAN SYNTHASE-LIKE8 (GSL8), a callose synthase that mediates the deposition of callose at developing cell plates, root hairs, and plasmodesmata. Unlike other gsl8 mutants, in which defects in cell plate formation are seedling lethal, cytokinetic defects in et2 predominantly occur in flowers and have little effect on vegetative growth and development. Similarly, mutations in STEROL METHYLTRANSFERASE2 (SMT2), a major sterol biosynthesis enzyme, also lead to weak cytokinetic defects, primarily in the flowers. In addition, SMT2 allelic mutants also generate tetraploid meiocytes through the ectopic induction of premeiotic endomitosis. These observations demonstrate that appropriate callose and sterol biosynthesis are required for maintaining the ploidy level of the premeiotic germ lineage and that subtle defects in cytokinesis may lead to diploid gametes and polyploid offspring.

SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana.

In meiosis, chromosome cohesion is maintained by the cohesin complex, which is released in a two-step manner. At meiosis I, the meiosis-specific cohesin subunit Rec8 is cleaved by the protease Separase along chromosome arms, allowing homologous chromosome segregation. Next, in meiosis II, cleavage of the remaining centromere cohesin results in separation of the sister chromatids. In eukaryotes, protection of centromeric cohesion in meiosis I is mediated by SHUGOSHINs (SGOs). The Arabidopsis genome contains two SGO homologs. Here we demonstrate that Atsgo1 mutants show a premature loss of cohesion of sister chromatid centromeres at anaphase I and that AtSGO2 partially rescues this loss of cohesion. In addition to SGOs, we characterize PATRONUS which is specifically required for the maintenance of cohesion of sister chromatid centromeres in meiosis II. In contrast to the Atsgo1 Atsgo2 double mutant, patronus T-DNA insertion mutants only display loss of sister chromatid cohesion after meiosis I, and additionally show disorganized spindles, resulting in defects in chromosome segregation in meiosis. This leads to reduced fertility and aneuploid offspring. Furthermore, we detect aneuploidy in sporophytic tissue, indicating a role for PATRONUS in chromosome segregation in somatic cells. Thus, ploidy stability is preserved in Arabidopsis by PATRONUS during both meiosis and mitosis.

Altered expression of Aurora kinases in Arabidopsis results in aneu- and polyploidization.

Aurora is an evolutionary conserved protein kinase family involved in monitoring of chromosome segregation via phosphorylation of different substrates. In plants, however, the involvement of Aurora proteins in meiosis and in sensing microtubule attachment remains to be proven, although the downstream components leading to the targeting of spindle assembly checkpoint signals to anaphase-promoting complex have been described. To analyze the three members of Aurora family (AtAurora1, -2, and -3) of Arabidopsis we employed different combinations of T-DNA insertion mutants and/or RNAi transformants. Meiotic defects and the formation of unreduced pollen were revealed including plants with an increased ploidy level. The effect of reduced expression of Aurora was mimicked by application of the ATP-competitive Aurora inhibitor II. In addition, strong overexpression of any member of the AtAurora family is not possible. Only tagged or truncated forms of Aurora kinases can be overexpressed. Expression of truncated AtAurora1 resulted in a high number of aneuploids in Arabidopsis, while expression of AtAurora1-TAPi construct in tobacco resulted in 4C (possible tetraploid) progeny. In conclusion, our data demonstrate an essential role of Aurora kinases in the monitoring of meiosis in plants.

The conserved chimeric transcript UPGRADE2 is associated with unreduced pollen formation and is exclusively found in apomictic Boechera species.

In apomictic Boechera spp., meiotic diplospory leads to the circumvention of meiosis and the suppression of recombination to produce unreduced male and female gametes (i.e. apomeiosis). Here, we have established an early flower developmental staging system and have performed microarray-based comparative gene expression analyses of the pollen mother cell stage in seven diploid sexual and seven diploid apomictic genotypes to identify candidate factors for unreduced pollen formation. We identified a transcript unique to apomictic Boechera spp. called UPGRADE2 (BspUPG2), which is highly up-regulated in their pollen mother cells. BspUPG2 is highly conserved among apomictic Boechera spp. genotypes but has no homolog in sexual Boechera spp. or in any other taxa. BspUPG2 undergoes posttranscriptional processing but lacks a prominent open reading frame. Together with the potential of stably forming microRNA-like secondary structures, we hypothesize that BspUPG2 functions as a long regulatory noncoding messenger RNA-like RNA. BspUPG2 has apparently arisen through a three-step process initiated by ancestral gene duplication of the original BspUPG1 locus, followed by sequential insertions of segmentally duplicated gene fragments, with final exonization of its sequence structure. Its genesis reflects the hybridization history that characterizes the genus Boechera.

The impact of environmental stress on male reproductive development in plants: biological processes and molecular mechanisms.

In plants, male reproductive development is extremely sensitive to adverse climatic environments and (a)biotic stress. Upon exposure to stress, male gametophytic organs often show morphological, structural and metabolic alterations that typically lead to meiotic defects or premature spore abortion and male reproductive sterility. Depending on the type of stress involved (e.g. heat, cold, drought) and the duration of stress exposure, the underlying cellular defect is highly variable and either involves cytoskeletal alterations, tapetal irregularities, altered sugar utilization, aberrations in auxin metabolism, accumulation of reactive oxygen species (ROS; oxidative stress) or the ectopic induction of programmed cell death (PCD). In this review, we present the critically stress-sensitive stages of male sporogenesis (meiosis) and male gametogenesis (microspore development), and discuss the corresponding biological processes involved and the resulting alterations in male reproduction. In addition, this review also provides insights into the molecular and/or hormonal regulation of the environmental stress sensitivity of male reproduction and outlines putative interaction(s) between the different processes involved.

Phenotyping, genetics, and "-omics" approaches to unravel and introgress enhanced resistance against apple scab (Venturia inaequalis) in apple cultivars (Malus × domestica).

Apple scab disease, caused by the fungus Venturia inaequalis, endangers commercial apple production globally. It is predominantly managed by frequent fungicide sprays that can harm the environment and promote the development of fungicide-resistant strains. Cultivation of scab-resistant cultivars harboring diverse qualitative Rvi resistance loci and quantitative trait loci associated with scab resistance could reduce the chemical footprint. A comprehensive understanding of the host-pathogen interaction is, however, needed to efficiently breed cultivars with enhanced resistance against a variety of pathogenic strains. Breeding efforts should not only encompass pyramiding of Rvi loci and their corresponding resistance alleles that directly or indirectly recognize pathogen effectors, but should also integrate genes that contribute to effective downstream defense mechanisms. This review provides an overview of the phenotypic and genetic aspects of apple scab resistance, and currently known corresponding defense mechanisms. Implementation of recent "-omics" approaches has provided insights into the complex network of physiological, molecular, and signaling processes that occur before and upon scab infection, thereby revealing the importance of both constitutive and induced defense mechanisms. Based on the current knowledge, we outline advances toward more efficient introgression of enhanced scab resistance into novel apple cultivars by conventional breeding or genetic modification techniques. However, additional studies integrating different "-omics" approaches combined with functional studies will be necessary to unravel effective defense mechanisms as well as key regulatory genes underpinning scab resistance in apple. This crucial information will set the stage for successful knowledge-based breeding for enhanced scab resistance.

CRISPR-Cas systems and applications for crop bioengineering.

CRISPR-Cas technologies contribute to enhancing our understanding of plant gene functions, and to the precise breeding of crop traits. Here, we review the latest progress in plant genome editing, focusing on emerging CRISPR-Cas systems, DNA-free delivery methods, and advanced editing approaches. By illustrating CRISPR-Cas applications for improving crop performance and food quality, we highlight the potential of genome-edited crops to contribute to sustainable agriculture and food security.

The influence of the pollination compatibility type on the pistil S-RNase expression in European pear (Pyrus communis).

The S-RNase gene plays an essential role in the gametophytic self-incompatibility (GSI) system of Pyrus. It codes for the stylar-expressed S-RNase protein which inhibits the growth of incompatible pollen tubes through cytotoxicity and the induction of programmed cell death in the pollen tube. While research on the Pyrus GSI system has primarily focused on the S-RNase gene, there is still a lack of insight into its spatiotemporal expression profile and the factors that regulate it. Previous studies have suggested that S-RNase expression in the style is influenced by pollination and is dependent on the compatibility type. We here continue on this basic hypothesis by analyzing the spatiotemporal expression of the S-RNase alleles in Pyrus communis "Conference" styles in response to different types of pollination; namely, upon full- and semi-compatible pollination and upon incompatible selfing. The results revealed that temporal dynamics of S-RNase expression are influenced by the pollen's compatibility type, indicating the presence of a signaling mechanism between pollen and style to control S-RNase production during pollen tube growth. In our experiment, S-RNase expression continuously decreased after cross-pollination and in the unpollinated control. However, after a fully incompatible pollination, S-RNase expression remained constant. Finally, semi-compatible pollination showed a initially constant S-RNase expression for both alleles followed by a strong decrease in expression. Based on these results and previous findings, we propose a regulatory mechanism to explain the effect of pollination and the associated compatibility type on S-RNase expression in the style. This proposed mechanism could be used as a starting point for future research.

Sexual polyploidization in plants--cytological mechanisms and molecular regulation.

In the plant kingdom, events of whole genome duplication or polyploidization are generally believed to occur via alterations of the sexual reproduction process. Thereby, diploid pollen and eggs are formed that contain the somatic number of chromosomes rather than the gametophytic number. By participating in fertilization, these so-called 2n gametes generate polyploid offspring and therefore constitute the basis for the establishment of polyploidy in plants. In addition, diplogamete formation, through meiotic restitution, is an essential component of apomixis and also serves as an important mechanism for the restoration of F1 hybrid fertility. Characterization of the cytological mechanisms and molecular factors underlying 2n gamete formation is therefore not only relevant for basic plant biology and evolution, but may also provide valuable cues for agricultural and biotechnological applications (e.g. reverse breeding, clonal seeds). Recent data have provided novel insights into the process of 2n pollen and egg formation and have revealed multiple means to the same end. Here, we summarize the cytological mechanisms and molecular regulatory networks underlying 2n gamete formation, and outline important mitotic and meiotic processes involved in the ectopic induction of sexual polyploidization.

Production of diploid male gametes in Arabidopsis by cold-induced destabilization of postmeiotic radial microtubule arrays.

Whole-genome duplication through the formation of diploid gametes is a major route for polyploidization, speciation, and diversification in plants. The prevalence of polyploids in adverse climates led us to hypothesize that abiotic stress conditions can induce or stimulate diploid gamete production. In this study, we show that short periods of cold stress induce the production of diploid and polyploid pollen in Arabidopsis (Arabidopsis thaliana). Using a combination of cytological and genetic analyses, we demonstrate that cold stress alters the formation of radial microtubule arrays at telophase II and consequently leads to defects in postmeiotic cytokinesis and cell wall formation. As a result, cold-stressed male meiosis generates triads, dyads, and monads that contain binuclear and polynuclear microspores. Fusion of nuclei in binuclear and polynuclear microspores occurs spontaneously before pollen mitosis I and eventually leads to the formation of diploid and polyploid pollen grains. Using segregation analyses, we also found that the majority of cold-induced dyads and triads are genetically equivalent to a second division restitution and produce diploid gametes that are highly homozygous. In a broader perspective, these findings offer insights into the fundamental mechanisms that regulate male gametogenesis in plants and demonstrate that their sensitivity to environmental stress has evolutionary significance and agronomic relevance in terms of polyploidization.

Pre-meiotic endomitosis in the cytokinesis-defective tomato mutant pmcd1 generates tetraploid meiocytes and diploid gametes.

Sexual polyploidization through the formation and functioning of 2n gametes is considered a major route for plant speciation and diversification. The cellular mechanism underlying 2n gamete formation mostly involves a restitution of the meiotic cell cycle, generating dyads and triads instead of tetrad meiotic end-products. As an alternative mechanism, the tomato mutant pmcd1 (for pre-meiotic cytokinesis defect 1), which generates diploid gametes through the ectopic induction of pre-meiotic endomitosis, is presented here. Using cytological approaches, it is demonstrated that male pmcd1 meiocyte initials exhibit clear alterations in cell cycle progression and cell plate formation, and consequently form syncytial cells that display different grades of cellular and/or nuclear fusion. In addition, it was found that other somatic tissue types (e.g. cotyledons and petals) also display occasional defects in cell wall formation and exhibit alterations in callose deposition, indicating that pmcd1 has a general defect in cell plate formation, most probably caused by alterations in callose biosynthesis. In a broader perspective, these findings demonstrate that defects in cytokinesis and cell plate formation may constitute a putative route for diplogamete formation and sexual polyploidization in plants.

Asynapsis and meiotic restitution in tomato male meiosis induced by heat stress.

Susceptibility of the reproductive system to temperature fluctuations is a recurrent problem for crop production under a changing climate. The damage is complex as multiple processes in male and female gamete formation are affected, but in general, particularly pollen production is impaired. Here, the impact of short periods of elevated temperature on male meiosis of tomato (Solanum lycopersicon L.) is reported. Meiocytes in early stage flower buds exposed to heat stress (>35°C) exhibit impaired homolog synapsis resulting in partial to complete omission of chiasmata formation. In the absence of chiasmata, univalents segregate randomly developing unbalanced tetrads and polyads resulting in aneuploid spores. However, most heat-stressed meiotic buds primarily contain balanced dyads, indicating a propensity to execute meiotic restitution. With most meiocytes exhibiting a complete loss of chiasma formation and concomitantly showing a mitotic-like division, heat stress triggers first division restitution resulting in clonal spores. These findings corroborate with the plasticity of male meiosis under heat and establish a natural route for the induction of sexual polyploidization in plants and the engineering of clonal seed.