ANNEXIN1 mediates calcium-dependent systemic defense in Arabidopsis plants upon herbivory and wounding.

Cellular calcium (Ca) transients are endogenous signals involved in local and systemic signaling and defense activation upon environmental stress, including wounding and herbivory. Still, not all Ca2+ channels contributing to the signaling have been identified, nor are their modes of action fully known. Plant annexins are proteins capable of binding to anionic phospholipids and can exhibit Ca channel-like activity. Arabidopsis ANNEXIN1 (ANN1) is suggested to contribute to Ca transport. Here, we report that wounding and simulated-herbivory-induced cytosolic free Ca elevation was impaired in systemic leaves in ann1 loss-of-function plants. We provide evidence for a role of ANN1 in local and systemic defense of plants attacked by herbivorous Spodoptera littoralis larvae. Bioassays identified ANN1 as a positive defense regulator. Spodoptera littoralis feeding on ann1 gained significantly more weight than larvae feeding on wild-type, whereas those feeding on ANN1-overexpressing lines gained less weight. Herbivory and wounding both induced defense-related responses on treated leaves, such as jasmonate accumulation and defense gene expression. These responses remained local and were strongly reduced in systemic leaves in ann1 plants. Our results indicate that ANN1 plays an important role in activation of systemic rather than local defense in plants attacked by herbivorous insects.

New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing.

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for "regenerated T0" lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase-ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.

Characterization of the nucellus-specific dehydrin MdoDHN11 demonstrates its involvement in the tolerance to water deficit.

KEY MESSAGE: MdoDHN11 acts in the nucellus layer to protect the embryo and the endosperm from limited water availability during apple seed development. Dehydrins (DHNs) are protective proteins related to several plant developmental responses that involve dehydration such as seed desiccation and abiotic stresses. In apple (Malus × domestica Borkh.), the seed-specific MdoDHN11 was suggested to play important roles against dehydration during seed development. However, this hypothesis has not yet been evaluated. Within this context, several experiments were performed to functionally characterize MdoDHN11. In situ hybridization analysis during apple seed development showed that MdoDHN11 expression is confined to a maternal tissue called nucellus, a central mass of parenchyma between the endosperm and the testa. The MdoDHN11 protein was localized in the cytosol and nucleus. Finally, transgenic Arabidopsis plants expressing MdoDHN11 were generated and exposed to a severe water-deficit stress, aiming to mimic a situation that can occurs during seed development. All transgenic lines showed increased tolerance to water deficit in relation to wild-type plants. Taken together, our results provide evidences that MdoDHN11 plays important roles during apple seed development by protecting the embryo and the endosperm from limited water availability, and the mechanism of action probably involves the interaction of MdoDHN11 with proteins and other components in the cell.

The MADS-box gene Agamous-like 11 is essential for seed morphogenesis in grapevine.

Despite the wide appreciation of seedless grapes, little is known about the molecular mechanisms that drive the stenospermocarpic seedless-type phenotype in grapevine. In order to address the molecular mechanisms that control seedlessness in grapevine, our study aimed to characterize VviAGL11, a class D MADS-box transcription factor gene that has been proposed as the major candidate gene involved in Vitis vinifera seed morphogenesis. VviAGL11 allelic variations in seeded and seedless grapevine cultivars were determined, and its correlations with allele-specific steady-state mRNA levels were investigated. VviAGL11 relative expression was significantly higher in seeds at 2, 4, and 6 weeks after fruit set, whereas in the seedless grape its transcript levels were extremely low in all stages analyzed. In situ hybridization revealed transcript accumulation specifically in the dual endotesta layer of the seeds, which is responsible for elongation and an increase of cell number, a necessary step to determine the lignification and the final seed size. No hybridization signals were visible in the seedless grapevine tissues, and a morphoanatomical analysis showed an apparent loss of identity of the endotesta layer of the seed traces. Ectopic expression of VviAGL11 in the Arabidopsis SEEDSTICK mutant background restored the wild-type phenotype and confirmed the direct role of VviAGL11 in seed morphogenesis, suggesting that depletion of its expression is responsible for the erroneous development of a highly essential seed layer, therefore culminating in the typical apirenic phenotype.

Chromatin Immunoprecipitation dataset of H3ac and H3K27me3 histone marks followed by DNA sequencing of Medicago truncatula embryos during control and heat stress conditions to decipher epigenetic regulation of desiccation tolerance acquisition.

Desiccation tolerance (DT) is one of the most important processes that seeds need to acquire during seed maturation because it will ensure survival until seeds have favourable conditions for germinating. Moreover, in the current climate warming context, heat stress and its impact on seed maturation and quality has been increasingly studied by the scientific community. Even if the transcriptomic changes enrolled in DT acquisition and seed heat stress response are fairly known, its epigenetic control has not yet been investigated. Medicago truncatula is a model legume for studying seed molecular mechanisms, which is known to display a delay in the acquisition of seed maturation mechanisms under heat conditions, except for desiccation acquisition. Our aim was to evaluate the role of two histone marks during embryo development under control and heat stress conditions on seed maturation processes, including the DT acquisition. These histone marks have either repressive (H3K27me3) or inducible (H3ac) effects on gene transcription, respectively corresponding to markers of packed and accessible chromatins. We identified all genomic regions bound to the H3K27me3 histones at four developmental stages and to the H3ac histones at the two earlier developmental stages during seed maturation, from seed filling to mature dry seeds, collected under optimal and heat stress conditions in the model legume, Medicago truncatula (reference genotype A17). A list of genes and promoters potentially linked to these two histone marks is reported and could provide clues about the epigenetic regulation of seed maturation between control and heat stress conditions, including the desiccation tolerance acquisition.

Chromatin dynamics associated with seed desiccation tolerance/sensitivity at early germination in Medicago truncatula.

Desiccation tolerance (DT) has contributed greatly to the adaptation of land plants to severe water-deficient conditions. DT is mostly observed in reproductive parts in flowering plants such as seeds. The seed DT is lost at early post germination stage but is temporally re-inducible in 1 mm radicles during the so-called DT window following a PEG treatment before being permanently silenced in 5 mm radicles of germinating seeds. The molecular mechanisms that activate/reactivate/silence DT in developing and germinating seeds have not yet been elucidated. Here, we analyzed chromatin dynamics related to re-inducibility of DT before and after the DT window at early germination in Medicago truncatula radicles to determine if DT-associated genes were transcriptionally regulated at the chromatin levels. Comparative transcriptome analysis of these radicles identified 948 genes as DT re-induction-related genes, positively correlated with DT re-induction. ATAC-Seq analyses revealed that the chromatin state of genomic regions containing these genes was clearly modulated by PEG treatment and affected by growth stages with opened chromatin in 1 mm radicles with PEG (R1P); intermediate openness in 1 mm radicles without PEG (R1); and condensed chromatin in 5 mm radicles without PEG (R5). In contrast, we also showed that the 103 genes negatively correlated with the re-induction of DT did not show any transcriptional regulation at the chromatin level. Additionally, ChIP-Seq analyses for repressive marks H2AK119ub and H3K27me3 detected a prominent signal of H3K27me3 on the DT re-induction-related gene sequences at R5 but not in R1 and R1P. Moreover, no clear H2AK119ub marks was observed on the DT re-induction-related gene sequences at both developmental radicle stages, suggesting that silencing of DT process after germination will be mainly due to H3K27me3 marks by the action of the PRC2 complex, without involvement of PRC1 complex. The dynamic of chromatin changes associated with H3K27me3 were also confirmed on seed-specific genes encoding potential DT-related proteins such as LEAs, oleosins and transcriptional factors. However, several transcriptional factors did not show a clear link between their decrease of chromatin openness and H3K27me3 levels, suggesting that their accessibility may also be regulated by additional factors, such as other histone modifications. Finally, in order to make these comprehensive genome-wide analyses of transcript and chromatin dynamics useful to the scientific community working on early germination and DT, we generated a dedicated genome browser containing all these data and publicly available at https://iris.angers.inrae.fr/mtseedepiatlas/jbrowse/?data=Mtruncatula.

Regulation of DNA (de)Methylation Positively Impacts Seed Germination during Seed Development under Heat Stress.

Seed development needs the coordination of multiple molecular mechanisms to promote correct tissue development, seed filling, and the acquisition of germination capacity, desiccation tolerance, longevity, and dormancy. Heat stress can negatively impact these processes and upon the increase of global mean temperatures, global food security is threatened. Here, we explored the impact of heat stress on seed physiology, morphology, gene expression, and methylation on three stages of seed development. Notably, Arabidopsis Col-0 plants under heat stress presented a decrease in germination capacity as well as a decrease in longevity. We observed that upon mild stress, gene expression and DNA methylation were moderately affected. Nevertheless, upon severe heat stress during seed development, gene expression was intensively modified, promoting heat stress response mechanisms including the activation of the ABA pathway. By analyzing candidate epigenetic markers using the mutants' physiological assays, we observed that the lack of DNA demethylation by the ROS1 gene impaired seed germination by affecting germination-related gene expression. On the other hand, we also observed that upon severe stress, a large proportion of differentially methylated regions (DMRs) were located in the promoters and gene sequences of germination-related genes. To conclude, our results indicate that DNA (de)methylation could be a key regulatory process to ensure proper seed germination of seeds produced under heat stress.

Medicago ABI3 Splicing Isoforms Regulate the Expression of Different Gene Clusters to Orchestrate Seed Maturation.

Seed maturation comprises important developmental processes, such as seed filling and the acquisition of seed germination capacity, desiccation tolerance, longevity, and dormancy. The molecular regulation of these processes is tightly controlled by the LAFL transcription factors, among which ABSCISIC ACID INSENSITIVE 3 (ABI3) was shown to be involved in most of these seed maturation processes. Here, we studied the ABI3 gene from Medicago truncatula, a model legume plant for seed studies. With the transcriptomes of two loss-of-function Medicago abi3 mutants, we were able to show that many gene classes were impacted by the abi3 mutation at different stages of early, middle, and late seed maturation. We also discovered three MtABI3 expression isoforms, which present contrasting expression patterns during seed development. Moreover, by ectopically expressing these isoforms in Medicago hairy roots generated from the abi3 mutant line background, we showed that each isoform regulated specific gene clusters, suggesting divergent molecular functions. Furthermore, we complemented the Arabidopsis abi3 mutant with each of the three MtABI3 isoforms and concluded that all isoforms were capable of restoring seed viability and desiccation tolerance phenotypes even if not all isoforms complemented the seed color phenotype. Taken together, our results allow a better understanding of the ABI3 network in Medicago during seed development, as well as the discovery of commonly regulated genes from the three MtABI3 isoforms, which can give us new insights into how desiccation tolerance and seed viability are regulated.

Manipulation of VviAGL11 expression changes the seed content in grapevine (Vitis vinifera L.).

Seedlessness in grapes is a desirable trait, especially for in natura consumption. Previously, we showed that VviAGL11 is the main responsible gene for seed morphogenesis in grapevine. Here we tested the function of this gene in grapevine with the use of plant plasmids. VviAGL11 was cloned into silencing and overexpression versions of p28iIR plasmid. Reproductive grapevine bunches from different seeded and seedless cultivars were separately treated with VviAGL11-harboring plasmids, along with controls. Plasmids were detected in leaves after a month of treatment, and berries, leaves, stems and seeds were analyzed for ectopic gene expression by RT-qPCR after 90 days of plasmid injection. Fruits from the seedless 'Linda' treated with the VviAGL11-overexpression plasmid showed high expression levels of VviAGL11 and exhibited small seeds that were not found in the untreated control samples. Mature grapes from seeded 'Italia' and 'Ruby' bunches treated with the VviAGL11-silencing plasmid showed decreased VviAGL11 expression, reduced number of seeds and increased number of seed traces. The present study confirms that VviAGL11 is a key master regulator of seed morphogenesis in grapevine and corroborates with the applicability of plant plasmids as promising biotechnological tools to functionally test genes in perennial plants in a rapid and confident way.

Evidence for GABA-Induced Systemic GABA Accumulation in Arabidopsis upon Wounding.

The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is present in all plant species analyzed so far. Its synthesis is stimulated by either acidic conditions occurring after tissue disruption or higher cytosolic calcium level. In mammals, GABA acts as inhibitory neurotransmitter but its function in plants is still not well understood. Besides its involvement in abiotic stress resistance, GABA has a role in the jasmonate-independent defense against invertebrate pests. While the biochemical basis for GABA accumulation in wounded leaves is obvious, the underlying mechanisms for wounding-induced GABA accumulation in systemic leaves remained unclear. Here, the Arabidopsis thaliana knock-out mutant lines pop2-5, unable to degrade GABA, and tpc1-2, lacking a wounding-induced systemic cytosolic calcium elevation, were employed for a comprehensive investigation of systemic GABA accumulation. A wounding-induced systemic GABA accumulation was detected in tpc1-2 plants demonstrating that an increased calcium level was not involved. Similarly, after both mechanical wounding and Spodoptera littoralis feeding, GABA accumulation in pop2-5 plants was significantly higher in local and systemic leaves, compared to wild-type plants. Consequently, larvae feeding on these GABA-enriched mutant plants grew significantly less. Upon exogenous application of a D2-labeled GABA to wounded leaves of pop2-5 plants, its uptake but no translocation to unwounded leaves was detected. In contrast, an accumulation of endogenous GABA was observed in vascular connected systemic leaves. These results suggest that the systemic accumulation of GABA upon wounding does not depend on the translocation of GABA or on an increase in cytosolic calcium.