Small molecule inhibitors of human LRRK2 enhance in vitro embryogenesis and microcallus formation for plant regeneration of crop and model species.

In vitro plant embryogenesis and microcallus formation are systems which are required for plant regeneration, a process during which cell reprogramming and proliferation are critical. These systems offer many advantages in breeding programmes, such as doubled-haploid production, clonal propagation of selected genotypes, and recovery of successfully gene-edited or transformed plants. However, the low proportion of reprogrammed cells in many plant species makes these processes highly inefficient. Here we report a new strategy to improve in vitro plant cell reprogramming using small molecule inhibitors of mammalian leucine rich repeat kinase 2 (LRRK2), which are used in pharmaceutical applications for cell reprogramming, but never used in plants before. LRRK2 inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of oilseed rape and barley, and somatic embryogenesis in cork oak. These inhibitors also promoted plant cell reprogramming and proliferation in Arabidopsis protoplast cultures. The benzothiazole derivative JZ1.24, a representative compound of the tested molecules, modified the expression of the brassinosteroid (BR)-related genes BIN2, CPD, and BAS1, correlating with an activation of BR signaling. Additionally, the LRRK2 inhibitor JZ1.24 induced the expression of the embryogenesis marker gene SERK1-like. The results suggest that the use of small molecules from the pharmaceutical field could be extended to promote in vitro reprogramming of plant cells towards embryogenesis or microcallus formation in a wider range of plant species and in vitro systems. This technological innovation would help to develop new strategies to improve the efficiency of in vitro plant regeneration, a major bottleneck in plant breeding.

Fibrin drives thromboinflammation and neuropathology in COVID-19.

Life-threatening thrombotic events and neurological symptoms are prevalent in COVID-19 and are persistent in patients with long COVID experiencing post-acute sequelae of SARS-CoV-2 infection1-4. Despite the clinical evidence1,5-7, the underlying mechanisms of coagulopathy in COVID-19 and its consequences in inflammation and neuropathology remain poorly understood and treatment options are insufficient. Fibrinogen, the central structural component of blood clots, is abundantly deposited in the lungs and brains of patients with COVID-19, correlates with disease severity and is a predictive biomarker for post-COVID-19 cognitive deficits1,5,8-10. Here we show that fibrin binds to the SARS-CoV-2 spike protein, forming proinflammatory blood clots that drive systemic thromboinflammation and neuropathology in COVID-19. Fibrin, acting through its inflammatory domain, is required for oxidative stress and macrophage activation in the lungs, whereas it suppresses natural killer cells, after SARS-CoV-2 infection. Fibrin promotes neuroinflammation and neuronal loss after infection, as well as innate immune activation in the brain and lungs independently of active infection. A monoclonal antibody targeting the inflammatory fibrin domain provides protection from microglial activation and neuronal injury, as well as from thromboinflammation in the lung after infection. Thus, fibrin drives inflammation and neuropathology in SARS-CoV-2 infection, and fibrin-targeting immunotherapy may represent a therapeutic intervention for patients with acute COVID-19 and long COVID.

Effects of cooling vest and personal protective equipment removal on thermoregulation in wildland firefighters during progressive thermal loads.

Background: Wildland firefighters (WFFs) regularly face demanding physical and environmental conditions during their duties, such as high ambient temperatures, challenging terrains, heavy equipment and protective gear. These conditions can strain thermoregulatory responses, leading to increased fatigue and posing risks to their health and safety. This study examined the effectiveness of two cooling interventions during physical activity in hot environments. Methods: Eight active male WFFs participated, comparing the effects of wearing a cooling vest (VEST) and personal protective equipment removal (PASSIVE) against a control condition (PPE). Participants walked on a treadmill at a speed of 6 km·h-1 for approximately 75-min under hot conditions (30°C and 30% relative humidity). Incremental slope increases were introduced every 15 min after the initial 20 min of activity, with 5-min passive recovery between each increment. Physiological and perceptual parameters were monitored throughout the protocol. Results: Significant main effects (p < 0.05) were observed in skin temperature (36.3 ± 0.2, 36.2 ± 0.4 and 35.4 ± 0.6°C in PPE, PASSIVE and VEST, respectively), physiological strain index (5.2 ± 0.4, 5.6 ± 1.1 and 4.3 ± 1.4 in PPE, PASSIVE and VEST) and thermal sensation (6.6 ± 0.6, 6.4 ± 0.7 and 5.3 ± 0.7 in PPE, PASSIVE, and VEST). However, no significant effects of the cooling strategies were observed on heart rate, gastrointestinal temperature or performance. Conclusion: Despite the observed effects on physiological responses, neither cooling strategy effectively mitigated thermal strain in WFFs under the experimental conditions tested.

Fibrinogen inhibits sonic hedgehog signaling and impairs neonatal cerebellar development after blood-brain barrier disruption.

Cerebellar injury in preterm infants with central nervous system (CNS) hemorrhage results in lasting neurological deficits and an increased risk of autism. The impact of blood-induced pathways on cerebellar development remains largely unknown, so no specific treatments have been developed to counteract the harmful effects of blood after neurovascular damage in preterm infants. Here, we show that fibrinogen, a blood-clotting protein, plays a central role in impairing neonatal cerebellar development. Longitudinal MRI of preterm infants revealed that cerebellar bleeds were the most critical factor associated with poor cerebellar growth. Using inflammatory and hemorrhagic mouse models of neonatal cerebellar injury, we found that fibrinogen increased innate immune activation and impeded neurogenesis in the developing cerebellum. Fibrinogen inhibited sonic hedgehog (SHH) signaling, the main mitogenic pathway in cerebellar granule neuron progenitors (CGNPs), and was sufficient to disrupt cerebellar growth. Genetic fibrinogen depletion attenuated neuroinflammation, promoted CGNP proliferation, and preserved normal cerebellar development after neurovascular damage. Our findings suggest that fibrinogen alters the balance of SHH signaling in the neurovascular niche and may serve as a therapeutic target to mitigate developmental brain injury after CNS hemorrhage.

Quality of life among Filipino breast cancer patients undergoing radiotherapy during the COVID-19 pandemic

Background@#The effect of treatment delays on the quality of life of breast cancer patients undergoing radiotherapy remains to be seen, especially from quarantine measures implemented during the COVID-19 pandemic.@*Objective@#The objective of this study is to assess the health-related quality of life (HR-QOL) of breast cancer patients referred for radiotherapy during the COVID-19 pandemic.@*Methodology@#This cross-sectional analysis included histopathologically proven breast cancer patients referred for radiotherapy at the Philippine General Hospital from June to October 2020. The University of the Philippines-Department of Health Quality of Life Scale for Cancer Patients was used to assess the HR-QOL of the respondents across five domains.@*Results@#A total of 60 respondents (median age of 52, range 33-71) were surveyed and eligible for analysis. College degree holders and good performers were associated with higher HR-QOL scores (p=0.008). The median interval from diagnosis to survey was 10.7 (SD±6.18) months and a longer illness duration was detrimental to HR-QOL. Overall, the global HR-QOL score was high (80.0% of respondents, HR-QOL score of 5.38±0.46). This was observed in all, except for the cognitive domain where HR-QOL was moderate among respondents (4.24±0.76).@*Conclusion@#This assessment was conducted within seven months into the pandemic, when an overall high HR-QOL score was observed among breast cancer patients. With further restrictions in treatment census encountered during the pandemic, strategies are recommended to address these indicators of health related QOL in this patient population through equitable and prompt access to needed care, such as radiotherapy.

Opposite Auxin Dynamics Determine the Gametophytic and Embryogenic Fates of the Microspore.

The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, in Brassica napus. We analyzed auxin concentration, cellular accumulation, the expression of the TAA1 auxin biosynthesis gene, and the PIN1-like efflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels and TAA1 and PIN1-like expression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis, TAA1 and PIN1-like genes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore.

Exploring the crop epigenome: a comparison of DNA methylation profiling techniques.

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

Defining blood-induced microglia functions in neurodegeneration through multiomic profiling.

Blood protein extravasation through a disrupted blood-brain barrier and innate immune activation are hallmarks of neurological diseases and emerging therapeutic targets. However, how blood proteins polarize innate immune cells remains largely unknown. Here, we established an unbiased blood-innate immunity multiomic and genetic loss-of-function pipeline to define the transcriptome and global phosphoproteome of blood-induced innate immune polarization and its role in microglia neurotoxicity. Blood induced widespread microglial transcriptional changes, including changes involving oxidative stress and neurodegenerative genes. Comparative functional multiomics showed that blood proteins induce distinct receptor-mediated transcriptional programs in microglia and macrophages, such as redox, type I interferon and lymphocyte recruitment. Deletion of the blood coagulation factor fibrinogen largely reversed blood-induced microglia neurodegenerative signatures. Genetic elimination of the fibrinogen-binding motif to CD11b in Alzheimer's disease mice reduced microglial lipid metabolism and neurodegenerative signatures that were shared with autoimmune-driven neuroinflammation in multiple sclerosis mice. Our data provide an interactive resource for investigation of the immunology of blood proteins that could support therapeutic targeting of microglia activation by immune and vascular signals.

Dynamics of Endogenous Auxin and Its Role in Somatic Embryogenesis Induction and Progression in Cork Oak.

Somatic embryogenesis (SE) is a feasible in vitro regeneration system with biotechnological applications in breeding programs, although, in many forest species, SE is highly inefficient, mainly due to their recalcitrance. On the other hand, SE represents a valuable model system for studies on cell reprogramming, totipotency acquisition, and embryogenic development. The molecular mechanisms that govern the transition of plant somatic cells to embryogenic cells are largely unknown. There is increasing evidence that auxins mediate this transition and play a key role in somatic embryo development, although data on woody species are very limited. In this study, we analyzed the dynamics and possible role of endogenous auxin during SE in cork oak (Quercus suber L.). The auxin content was low in somatic cells before cell reprogramming, while it increased after induction of embryogenesis, as revealed by immunofluorescence assays. Cellular accumulation of endogenous auxin was also detected at the later stages of somatic embryo development. These changes in auxin levels correlated with the expression patterns of the auxin biosynthesis (QsTAR2) and signaling (QsARF5) genes, which were upregulated after SE induction. Treatments with the inhibitor of auxin biosynthesis, kynurenine, reduced the proliferation of proembryogenic masses and impaired further embryo development. QsTAR2 and QsARF5 were downregulated after kynurenine treatment. Our findings indicate a key role of endogenous auxin biosynthesis and signaling in SE induction and multiplication, as well as somatic embryo development of cork oak.

The wild plant Gnaphalium lavandulifolium as a sentinel for biomonitoring the effects of environmental heavy metals in the metropolitan area of México Valley.

Biomonitoring is a valuable tool for assessing the presence and effects of air pollutants such as heavy metals (HM); due to their toxicity and stability, these compounds can affect human health and the balance of ecosystems. To assess its potential as a sentinel organism of HM pollution, the wild plant Gnaphalium lavandulifolium was exposed to four sites in the metropolitan area of México Valley (MAMV): Altzomoni (ALT) Coyoacán (COY), Ecatepec (ECA), and Tlalnepantla (TLA) during 2, 4, and 8 weeks, between October and November 2019. Control plants remained under controlled conditions. The chemical analysis determined twelve HM (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn) in the leaves. Macroscopic damage to the leaves, later determined in semi-thin sections under light microscopy, lead to a finer analysis. Transmission electron microscope (TEM) showed major structural changes: chromatin condensation, protoplast shrinkage, cytoplasm vacuolization, cell wall thinning, decreased number and size of starch grains, and plastoglobules in chloroplasts. All these characteristics of stress-induced programed cell death (sPCD) were related to the significant increase of toxic HM in the leaves of the exposed plants compared to the control (p < 0.05). Immunohistochemistry revealed a significant amount of proteases with caspase 3-like activity in ECA and TLA samples during long exposure times. Ultrastructural changes and sPCD features detected confirmed the usefulness of G. lavandulifolium as a good biomonitor of HM contamination. They supported the possibility of considering subcellular changes as markers of abiotic stress conditions in plants.

Effects of Citrulline Supplementation on Different Aerobic Exercise Performance Outcomes: A Systematic Review and Meta-Analysis.

Supplementation with Citrulline (Cit) has been shown to have a positive impact on aerobic exercise performance and related outcomes such as lactate, oxygen uptake (VO2) kinetics, and the rate of perceived exertion (RPE), probably due to its relationship to endogenous nitric oxide production. However, current research has shown this to be controversial. The main objective of this systematic review and meta-analysis was to analyze and assess the effects of Cit supplementation on aerobic exercise performance and related outcomes, as well as to show the most suitable doses and timing of ingestion. A structured literature search was carried out by the PRISMA® (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) and PICOS guidelines in the following databases: Pubmed/Medline, Scopus, and Web of Science (WOS). A total of 10 studies were included in the analysis, all of which exclusively compared the effects of Cit supplementation with those of a placebo group on aerobic performance, lactate, VO2, and the RPE. Those articles that used other supplements and measured other outcomes were excluded. The meta-analysis was carried out using Hedges' g random effects model and pooled standardized mean differences (SMD). The results showed no positive effects of Cit supplementation on aerobic performance (pooled SMD = 0.15; 95% CI (-0.02 to 0.32); I2, 0%; p = 0.08), the RPE (pooled SMD = -0.03; 95% CI (-0.43 to 0.38); I2, 49%; p = 0.9), VO2 kinetics (pooled SMD = 0.01; 95% CI (-0.16 to 0.17); I2, 0%; p = 0.94), and lactate (pooled SMD = 0.25; 95% CI (-0.10 to 0.59); I2, 0%; p = 0.16). In conclusion, Cit supplementation did not prove to have any benefits for aerobic exercise performance and related outcomes. Where chronic protocols seemed to show a positive tendency, more studies in the field are needed to better understand the effects.

The DNA methylation landscape of the root-knot nematode-induced pseudo-organ, the gall, in Arabidopsis, is dynamic, contrasting over time, and critically important for successful parasitism.

Root-knot nematodes (RKNs) induce giant cells (GCs) within galls which are characterized by large-scale gene repression at early stages. However, the epigenetic mechanism(s) underlying gene silencing is (are) still poorly characterized. DNA methylation in Arabidopsis galls induced by Meloidogyne javanica was studied at crucial infection stages (3 d post-infection (dpi) and 14 dpi) using enzymatic, cytological, and sequencing approaches. DNA methyltransferase mutants (met1, cmt2, cmt3, cmt2/3, drm1/2, ddc) and a DNA demethylase mutant (ros1), were analyzed for RKN resistance/tolerance, and galls were characterized by confocal microscopy and RNA-seq. Early galls were hypermethylated, and the GCs were found to be the major contributors to this hypermethylation, consistent with the very high degree of gene repression they exhibit. By contrast, medium/late galls showed no global increase in DNA methylation compared to uninfected roots, but exhibited large-scale redistribution of differentially methylated regions (DMRs). In line with these findings, it was also shown that DNA methylation and demethylation mutants showed impaired nematode reproduction and gall/GC-development. Moreover, siRNAs that were exclusively present in early galls accumulated at hypermethylated DMRs, overlapping mostly with retrotransposons in the CHG/CG contexts that might be involved in their repression, contributing to their stability/genome integrity. Promoter/gene methylation correlated with differentially expressed genes encoding proteins with basic cell functions. Both mechanisms are consistent with reprogramming host tissues for gall/GC formation. In conclusion, RNA-directed DNA methylation (RdDM; DRM2/1) pathways, maintenance methyltransferases (MET1/CMT3) and demethylation (ROS1) appear to be prominent mechanisms driving a dynamic regulation of the epigenetic landscape during RKN infection.

Epigenetics for Crop Improvement in Times of Global Change.

Epigenetics has emerged as an important research field for crop improvement under the on-going climatic changes. Heritable epigenetic changes can arise independently of DNA sequence alterations and have been associated with altered gene expression and transmitted phenotypic variation. By modulating plant development and physiological responses to environmental conditions, epigenetic diversity-naturally, genetically, chemically, or environmentally induced-can help optimise crop traits in an era challenged by global climate change. Beyond DNA sequence variation, the epigenetic modifications may contribute to breeding by providing useful markers and allowing the use of epigenome diversity to predict plant performance and increase final crop production. Given the difficulties in transferring the knowledge of the epigenetic mechanisms from model plants to crops, various strategies have emerged. Among those strategies are modelling frameworks dedicated to predicting epigenetically controlled-adaptive traits, the use of epigenetics for in vitro regeneration to accelerate crop breeding, and changes of specific epigenetic marks that modulate gene expression of traits of interest. The key challenge that agriculture faces in the 21st century is to increase crop production by speeding up the breeding of resilient crop species. Therefore, epigenetics provides fundamental molecular information with potential direct applications in crop enhancement, tolerance, and adaptation within the context of climate change.

Small molecule inhibitors of mammalian GSK-3ß promote in vitro plant cell reprogramming and somatic embryogenesis in crop and forest species.

Plant in vitro regeneration systems, such as somatic embryogenesis, are essential in breeding; they permit propagation of elite genotypes, production of doubled-haploids, and regeneration of whole plants from gene editing or transformation events. However, in many crop and forest species, somatic embryogenesis is highly inefficient. We report a new strategy to improve in vitro embryogenesis using synthetic small molecule inhibitors of mammalian glycogen synthase kinase 3ß (GSK-3ß), never used in plants. These inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of Brassica napus and Hordeum vulgare, and somatic embryogenesis of Quercus suber. TDZD-8, a representative compound of the molecules tested, inhibited GSK-3 activity in microspore cultures, and increased expression of embryogenesis genes FUS3, LEC2, and AGL15. Plant GSK-3 kinase BIN2 is a master regulator of brassinosteroid (BR) signalling. During microspore embryogenesis, BR biosynthesis and signalling genes CPD, GSK-3-BIN2, BES1, and BZR1 were up-regulated and the BAS1 catabolic gene was repressed, indicating activation of the BR pathway. TDZD-8 increased expression of BR signalling elements, mimicking BR effects. The findings support that the small molecule inhibitors promoted somatic embryogenesis by activating the BR pathway, opening up the way for new strategies using GSK-3ß inhibitors that could be extended to other species.

Body Composition, Dietary Intake and the Risk of Low Energy Availability in Elite-Level Competitive Rhythmic Gymnasts.

The aim of this study was to analyze dietary intake and body composition in a group of elite-level competitive rhythmic gymnasts from Spain. We undertook body composition and nutritional analysis of 30 elite gymnasts, divided into two groups by age: pre-teen (9-12 years) (n = 17) and teen (13-18 years) (n = 13). Measures of height, weight, and bioimpedance were used to calculate body mass index and percent body fat. Energy and nutrient intakes were assessed based on 7-day food records. The two groups had similar percentages of total body fat (pre-teen: 13.99 ± 3.83% vs. teen: 14.33 ± 5.57%; p > 0.05). The energy availability values for pre-teens were above the recommended values (>40 kcal/FFM/day) 69.38 ± 14.47 kcal/FFM/day, while those for the teens were much lower (34.7 ± 7.5 kcal/FFM/day). The distribution of the daily energy intake across the macronutrients indicates that both groups ingested less than the recommended level of carbohydrates and more than the recommended level of fat. Very low intakes of calcium and vitamin D among other micronutrients were also noted. The main finding is that teenage gymnasts do not consume as much energy as they need each day, which explains their weight and development. Moreover, they are at a high risk of developing low energy availability that could negatively impact their performance and future health.

Deciphering the Epigenetic Alphabet Involved in Transgenerational Stress Memory in Crops.

Although epigenetic modifications have been intensely investigated over the last decade due to their role in crop adaptation to rapid climate change, it is unclear which epigenetic changes are heritable and therefore transmitted to their progeny. The identification of epigenetic marks that are transmitted to the next generations is of primary importance for their use in breeding and for the development of new cultivars with a broad-spectrum of tolerance/resistance to abiotic and biotic stresses. In this review, we discuss general aspects of plant responses to environmental stresses and provide an overview of recent findings on the role of transgenerational epigenetic modifications in crops. In addition, we take the opportunity to describe the aims of EPI-CATCH, an international COST action consortium composed by researchers from 28 countries. The aim of this COST action launched in 2020 is: (1) to define standardized pipelines and methods used in the study of epigenetic mechanisms in plants, (2) update, share, and exchange findings in epigenetic responses to environmental stresses in plants, (3) develop new concepts and frontiers in plant epigenetics and epigenomics, (4) enhance dissemination, communication, and transfer of knowledge in plant epigenetics and epigenomics.

Endogenous auxin accumulation/localization during zygotic and somatic embryogenesis of Capsicum chinense Jacq.

Zygotic and somatic embryogenesis in plants is a fascinating event that is finely regulated through the expression of a specific group of genes and dynamic levels of plant hormones whose concerted action determines the fate that specific cells follow towards zygotic or somatic embryo development. This work studied different stages of Capsicum chinense Jacq. zygotic and somatic embryogenesis. HPLC quantification determined that the levels of indole-3-acetic acid (IAA) increase as the zygotic or somatic embryogenesis progresses, being higher at maturity, thus supporting a positive correlation between embryo cell differentiation and IAA increase. A monoclonal anti-IAA-antibody was used to detect IAA levels. Findings revealed a dynamic pattern of auxin distribution along the different embryogenic embryonic stages. In the early stages of zygotic embryos, the IAA gradient was observed in the basal cells of the suspensor and the hypostases, suggesting that they are the initial source of the IAA hormone. As embryogenesis proceeds, the dynamic of the IAA gradient is displaced to the embryo and endosperm cells. In the case of induced somatic embryogenesis, the IAA gradient was detected in the dividing cells of the endodermis, from where pre-embryogenic cells emerge. However, the analysis of somatic embryos revealed that IAA was homogeneously distributed. This study shows evidence supporting a correlation between IAA levels during zygotic or somatic embryogenesis in Capsicum chinense species.

Suppression of Metacaspase- and Autophagy-Dependent Cell Death Improves Stress-Induced Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a biotechnological process that allows us to rapidly obtain doubled-haploid plants for breeding programs. The process is initiated by the application of stress treatment, which reprograms microspores to embark on embryonic development. Typically, a part of the microspores undergoes cell death that reduces the efficiency of the process. Metacaspases (MCAs), a phylogenetically broad group of cysteine proteases, and autophagy, the major catabolic process in eukaryotes, are critical regulators of the balance between cell death and survival in various organisms. In this study, we analyzed the role of MCAs and autophagy in cell death during stress-induced microspore embryogenesis in Brassica napus. We demonstrate that this cell death is accompanied by the transcriptional upregulation of three BnMCA genes (BnMCA-Ia, BnMCA-IIa and BnMCA-IIi), an increase in MCA proteolytic activity and the activation of autophagy. Accordingly, inhibition of autophagy and MCA activity, either individually or in combination, suppressed cell death and increased the number of proembryos, indicating that both components play a pro-cell death role and account for decreased efficiency of early embryonic development. Therefore, MCAs and/or autophagy can be used as new biotechnological targets to improve in vitro embryogenesis in Brassica species and doubled-haploid plant production in crop breeding and propagation programs.