Heterologous expression of a lycophyte protein enhances angiosperm seedling vigor.

Seedling vigor is a key agronomic trait that determines juvenile plant performance. Angiosperm seeds develop inside fruits and are connected to the mother plant through vascular tissues. Their formation requires plant-specific genes, such as BREVIS RADIX (BRX) in Arabidopsis thaliana roots. BRX family proteins are found throughout the euphyllophytes but also occur in non-vascular bryophytes and non-seed lycophytes. They consist of four conserved domains, including the tandem BRX domains. We found that bryophyte or lycophyte BRX homologs can only partially substitute for Arabidopsis BRX (AtBRX) because they miss key features in the linker between the BRX domains. Intriguingly, however, expression of a BRX homolog from the lycophyte Selaginella moellendorffii (SmBRX) in an A. thaliana wild-type background confers robustly enhanced root growth vigor that persists throughout the life cycle. This effect can be traced to a substantial increase in seed and embryo size, is associated with enhanced vascular tissue proliferation, and can be reproduced with a modified, SmBRX-like variant of AtBRX. Our results thus suggest that BRX variants can boost seedling vigor and shed light on the activity of ancient, non-angiosperm BRX family proteins.

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.

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.

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.

Modulation of autophagy and protease activities by small bioactive compounds to reduce cell death and improve stress-induced microspore embryogenesis initiation in rapeseed and barley.

Microspore embryogenesis is a powerful biotechnological tool that is very useful in crop breeding for the rapid production of haploid and double-haploid embryos and plants. In this in vitro system, the haploid microspore is reprogrammed by the application of specific stress treatments. A high level of cell death after the stress is a major factor that greatly reduces embryogenesis yield at its initial stages. Autophagy is a degradation pathway that is present in all eukaryotes and plays key roles in a range of processes, including stress responses. Many proteases participate in autophagy and cell death; among them, cathepsins are the most abundant enzymes with a role in plant senescence and programmed cell death (PCD). Moreover, although plant genomes do not contain homologues of caspases, caspase 3-like activity (main executioner protease of animal cell death) has been detected in many plant PCD processes. Recent studies by our group in barley microspore cultures reported that the stress treatment required for inducing microspore embryogenesis (cold treatment), also produced reactive oxygen species (ROS) and cell death, concomitantly with the induction of autophagy, as well as cathepsin-like and caspase 3-like proteolytic activities. In the present study, we report new data on microspore embryogenesis of rapeseed that indicate, as in barley, activation of cell death and autophagy processes after the inductive stress. The results revealed that treatments modulating autophagy and proteases produced the same effect in the two plant systems, regardless of the stress applied, cold in barley or heat in rapeseed. Pharmacological treatments with small bioactive compounds that inhibit ROS, autophagy and specific cell death-proteases led to reduced cell death and an increased embryogenesis initiation rate in both, barley and rapeseed. Taken together, these findings open up new intervention pathways by modulating autophagy and proteases, which are very promising in terms of increasing the efficiency of in vitro microspore embryogenesis systems for biotechnological applications in crop breeding.

Autophagy is activated and involved in cell death with participation of cathepsins during stress-induced microspore embryogenesis in barley.

Microspores are reprogrammed towards embryogenesis by stress. Many microspores die after this stress, limiting the efficiency of microspore embryogenesis. Autophagy is a degradation pathway that plays critical roles in stress response and cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; less is known in plants. Plant cathepsins are papain-like C1A cysteine proteases involved in many physiological processes, including programmed cell death. We have analysed the involvement of autophagy in cell death, in relation to cathepsin activation, during stress-induced microspore embryogenesis in Hordeum vulgare. After stress, reactive oxygen species (ROS) and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8, and autophagosomes. Concomitantly, cathepsin L/F-, B-, and H-like activities were induced, cathepsin-like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6, and HvPap-19 proteins increased and localized in the cytoplasm, resembling autophagy structures. Inhibitors of autophagy and cysteine proteases reduced cell death and promoted embryogenesis. The findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, and the participation of cathepsins. Similar patterns of activation, expression, and localization suggest a possible connection between cathepsins and autophagy. The results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine protease modulators.

Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber.

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with ß-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

Inhibition of Histone H3K9 Methylation by BIX-01294 Promotes Stress-Induced Microspore Totipotency and Enhances Embryogenesis Initiation.

Microspore embryogenesis is a process of cell reprogramming, totipotency acquisition and embryogenesis initiation, induced in vitro by stress treatments and widely used in plant breeding for rapid production of doubled-haploids, but its regulating mechanisms are still largely unknown. Increasing evidence has revealed epigenetic reprogramming during microspore embryogenesis, through DNA methylation, but less is known about the involvement of histone modifications. In this study, we have analyzed the dynamics and possible role of histone H3K9 methylation, a major repressive modification, as well as the effects on microspore embryogenesis initiation of BIX-01294, an inhibitor of histone methylation, tested for the first time in plants, in Brassica napus and Hordeum vulgare. Results revealed that microspore reprogramming and initiation of embryogenesis involved a low level of H3K9 methylation. With the progression of embryogenesis, methylation of H3K9 increased, correlating with gene expression profiles of BnHKMT SUVR4-like and BnLSD1-like (writer and eraser enzymes of H3K9me2). At early stages, BIX-01294 promoted cell reprogramming, totipotency and embryogenesis induction, while diminishing bulk H3K9 methylation. DNA methylation was also reduced by short-term BIX-01294 treatment. By contrast, long BIX-01294 treatments hindered embryogenesis progression, indicating that H3K9 methylation is required for embryo differentiation. These findings open up new possibilities to enhance microspore embryogenesis efficiency in recalcitrant species through pharmacological modulation of histone methylation by using BIX-01294.

BnPME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus.

BACKGROUND: Pectins are one of the main components of plant cell walls. They are secreted to the wall as highly methylesterified forms that can be de-esterified by pectin methylesterases (PMEs). The degree of methylesterification of pectins changes during development, PMEs are involved in the cell wall remodeling that occurs during diverse plant developmental processes. Nevertheless, the functional meaning of pectin-related wall remodeling in different cell types and processes remains unclear. In vivo, the microspore follows the gametophytic pathway and differentiates to form the pollen grain. In vitro, the microspore can be reprogrammed by stress treatments becoming a totipotent cell that starts to proliferate and follows the embryogenic pathway, a process known as microspore embryogenesis. RESULTS: To investigate if the change of developmental programme of the microspore towards embryogenesis involves changes in pectin esterification levels, which would cause the cell wall remodeling during the process, in the present study, dynamics of PME expression and degrees of pectin esterification have been analysed during microspore embryogenesis and compared with the gametophytic development, in Brassica napus. A multidisciplinary approach has been adopted including BnPME gene expression analysis by quantitative RT-PCR, fluorescence in situ hybridization, immuno-dot-blot and immunofluorescence with JIM5 and JIM7 antibodies to reveal low and highly-methylesterified pectins. The results showed that cell differentiation at advanced developmental stages involved induction of BnPME expression and pectin de-esterification, processes that were also detected in zygotic embryos, providing additional evidence that microspore embryogenesis mimics zygotic embryogenesis. By contrast, early microspore embryogenesis, totipotency and proliferation were associated with low expression of BnPME and high levels of esterified pectins. CONCLUSIONS: The results show that the change of developmental programme of the microspore involves changes in pectin esterification associated with proliferation and differentiation events, which may cause the cell wall remodeling during the process. The findings indicate pectin-related modifications in the cell wall during microspore embryogenesis, providing new insights into the role of pectin esterification and cell wall configuration in microspore totipotency, embryogenesis induction and progression.