The plastidial protein acetyltransferase GNAT1 forms a complex with GNAT2, yet their interaction is dispensable for state transitions.

Protein N-acetylation is one of the most abundant co- and post-translational modifications in eukaryotes, extending its occurrence to chloroplasts within vascular plants. Recently, a novel plastidial enzyme family comprising eight acetyltransferases that exhibit dual lysine and N-terminus acetylation activities was unveiled in Arabidopsis. Among these, GNAT1, GNAT2, and GNAT3 reveal notable phylogenetic proximity, forming a subgroup termed NAA90. Our study focused on characterizing GNAT1, closely related to the state transition acetyltransferase GNAT2. In contrast to GNAT2, GNAT1 did not prove essential for state transitions and displayed no discernible phenotypic difference compared to the wild type under high light conditions, while gnat2 mutants were severely affected. However, gnat1 mutants exhibited a tighter packing of the thylakoid membranes akin to gnat2 mutants. In vitro studies with recombinant GNAT1 demonstrated robust N-terminus acetylation activity on synthetic substrate peptides. This activity was confirmed in vivo through N-terminal acetylome profiling in two independent gnat1 knockout lines. This attributed several acetylation sites on plastidial proteins to GNAT1, reflecting a subset of GNAT2's substrate spectrum. Moreover, co-immunoprecipitation coupled to mass spectrometry revealed a robust interaction between GNAT1 and GNAT2, as well as a significant association of GNAT2 with GNAT3 - the third acetyltransferase within the NAA90 subfamily. This study unveils the existence of at least two acetyltransferase complexes within chloroplasts, whereby complex formation might have a critical effect on the fine-tuning of the overall acetyltransferase activities. These findings introduce a novel layer of regulation in acetylation-dependent adjustments in plastidial metabolism.

ß-Glucan-binding proteins are key modulators of immunity and symbiosis in mutualistic plant-microbe interactions.

In order to discriminate between detrimental, commensal, and beneficial microbes, plants rely on polysaccharides such as ß-glucans, which are integral components of microbial and plant cell walls. The conversion of cell wall-associated ß-glucan polymers into a specific outcome that affects plant-microbe interactions is mediated by hydrolytic and non-hydrolytic ß-glucan-binding proteins. These proteins play crucial roles during microbial colonization: they influence the composition and resilience of host and microbial cell walls, regulate the homeostasis of apoplastic concentrations of ß-glucan oligomers, and mediate ß-glucan perception and signaling. This review outlines the dual roles of ß-glucans and their binding proteins in plant immunity and symbiosis, highlighting recent discoveries on the role of ß-glucan-binding proteins as modulators of immunity and as symbiosis receptors involved in the fine-tuning of microbial accommodation.

Amphicarpic development in Cardamine chenopodiifolia.

Amphicarpy is an unusual trait where two fruit types develop on the same plant: one above and the other belowground. This trait is not found in conventional model species. Therefore, its development and molecular genetics remain under-studied. Here, we establish the allooctoploid Cardamine chenopodiifolia as an emerging experimental system to study amphicarpy. We characterized C. chenopodiifolia development, focusing on differences in morphology and cell wall histochemistry between above- and belowground fruit. We generated a reference transcriptome with PacBio full-length transcript sequencing and analysed differential gene expression between above- and belowground fruit valves. Cardamine chenopodiifolia has two contrasting modes of seed dispersal. The main shoot fails to bolt and initiates floral primordia that grow underground where they self-pollinate and set seed. By contrast, axillary shoots bolt and develop exploding seed pods aboveground. Morphological differences between aerial explosive fruit and subterranean nonexplosive fruit were reflected in a large number of differentially regulated genes involved in photosynthesis, secondary cell wall formation and defence responses. Tools established in C. chenopodiifolia, such as a reference transcriptome, draft genome assembly and stable plant transformation, pave the way to study amphicarpy and trait evolution via allopolyploidy.

The Arabidopsis SGN3/GSO1 receptor kinase integrates soil nitrogen status into shoot development.

The Casparian strip is a barrier in the endodermal cell walls of plants that allows the selective uptake of nutrients and water. In the model plant Arabidopsis thaliana, its development and establishment are under the control of a receptor-ligand mechanism termed the Schengen pathway. This pathway facilitates barrier formation and activates downstream compensatory responses in case of dysfunction. However, due to a very tight functional association with the Casparian strip, other potential signaling functions of the Schengen pathway remain obscure. In this work, we created a MYB36-dependent synthetic positive feedback loop that drives Casparian strip formation independently of Schengen-induced signaling. We evaluated this by subjecting plants in which the Schengen pathway has been uncoupled from barrier formation, as well as a number of established barrier-mutant plants, to agar-based and soil conditions that mimic agricultural settings. Under the latter conditions, the Schengen pathway is necessary for the establishment of nitrogen-deficiency responses in shoots. These data highlight Schengen signaling as an essential hub for the adaptive integration of signaling from the rhizosphere to aboveground tissues.

The development of pleiotropic phenotypes in powdery mildew-resistant barley and Arabidopsis thaliana mlo mutants is linked to nitrogen availability.

Powdery mildew-resistant barley (Hordeum vulgare) and Arabidopsis thaliana mlo mutant plants exhibit pleiotropic phenotypes such as the spontaneous formation of callose-rich cell wall appositions and early leaf chlorosis and necrosis, indicative of premature leaf senescence. The exogenous factors governing the occurrence of these undesired side effects remain poorly understood. Here, we characterised the formation of these symptoms in detail. Ultrastructural analysis revealed that the callose-rich cell wall depositions spontaneously formed in A. thaliana mlo mutants are indistinguishable from those induced by the bacterial pattern epitope, flagellin 22 (flg22). We further found that increased plant densities during culturing enhance the extent of the leaf senescence syndrome in A. thaliana mlo mutants. Application of a liquid fertiliser rescued the occurrence of leaf chlorosis and necrosis in both A. thaliana and barley mlo mutant plants. Controlled fertilisation experiments uncovered nitrogen as the macronutrient whose deficiency promotes the extent of pleiotropic phenotypes in A. thaliana mlo mutants. Light intensity and temperature had a modulatory impact on the incidence of leaf necrosis in the case of barley mlo mutant plants. Collectively, our data indicate that the development of pleiotropic phenotypes associated with mlo mutants is governed by various exogenous factors.

Cell-cycle-linked growth reprogramming encodes developmental time into leaf morphogenesis.

How is time encoded into organ growth and morphogenesis? We address this question by investigating heteroblasty, where leaf development and form are modified with progressing plant age. By combining morphometric analyses, fate-mapping through live-imaging, computational analyses, and genetics, we identify age-dependent changes in cell-cycle-associated growth and histogenesis that underpin leaf heteroblasty. We show that in juvenile leaves, cell proliferation competence is rapidly released in a "proliferation burst" coupled with fast growth, whereas in adult leaves, proliferative growth is sustained for longer and at a slower rate. These effects are mediated by the SPL9 transcription factor in response to inputs from both shoot age and individual leaf maturation along the proximodistal axis. SPL9 acts by activating CyclinD3 family genes, which are sufficient to bypass the requirement for SPL9 in the control of leaf shape and in heteroblastic reprogramming of cellular growth. In conclusion, we have identified a mechanism that bridges across cell, tissue, and whole-organism scales by linking cell-cycle-associated growth control to age-dependent changes in organ geometry.

Three-Dimensional Ultrastructure of Arabidopsis Cotyledons Infected with Colletotrichum higginsianum.

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host-pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum. By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum-Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral (x-y) pixel resolution of 10 nm and an axial (z) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum-Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

A high-confidence Physcomitrium patens plasmodesmata proteome by iterative scoring and validation reveals diversification of cell wall proteins during evolution.

Plasmodesmata (PD) facilitate movement of molecules between plant cells. Regulation of this movement is still not understood. Plasmodesmata are hard to study, being deeply embedded within cell walls and incorporating several membrane types. Thus, structure and protein composition of PD remain enigmatic. Previous studies of PD protein composition identified protein lists with few validations, making functional conclusions difficult. We developed a PD scoring approach in iteration with large-scale systematic localization, defining a high-confidence PD proteome of Physcomitrium patens (HC300). HC300, together with bona fide PD proteins from literature, were placed in Pddb. About 65% of proteins in HC300 were not previously PD-localized. Callose-degrading glycolyl hydrolase family 17 (GHL17) is an abundant protein family with representatives across evolutionary scale. Among GHL17s, we exclusively found members of one phylogenetic clade with PD localization and orthologs occur only in species with developed PD. Phylogenetic comparison was expanded to xyloglucan endotransglucosylases/hydrolases and Exordium-like proteins, which also diversified into PD-localized and non-PD-localized members on distinct phylogenetic clades. Our high-confidence PD proteome HC300 provides insights into diversification of large protein families. Iterative and systematic large-scale localization across plant species strengthens the reliability of HC300 as basis for exploring structure, function, and evolution of this important organelle.

A conserved enzyme of smut fungi facilitates cell-to-cell extension in the plant bundle sheath.

Smut fungi comprise one of the largest groups of fungal plant pathogens causing disease in all cereal crops. They directly penetrate host tissues and establish a biotrophic interaction. To do so, smut fungi secrete a wide range of effector proteins, which suppress plant immunity and modulate cellular functions as well as development of the host, thereby determining the pathogen's lifestyle and virulence potential. The conserved effector Erc1 (enzyme required for cell-to-cell extension) contributes to virulence of the corn smut Ustilago maydis in maize leaves but not on the tassel. Erc1 binds to host cell wall components and displays 1,3-ß-glucanase activity, which is required to attenuate ß-glucan-induced defense responses. Here we show that Erc1 has a cell type-specific virulence function, being necessary for fungal cell-to-cell extension in the plant bundle sheath and this function is fully conserved in the Erc1 orthologue of the barley pathogen Ustilago hordei.

FLOWERING REPRESSOR AAA+ ATPase 1 is a novel regulator of perennial flowering in Arabis alpina.

Arabis alpina is a polycarpic perennial, in which PERPETUAL FLOWERING1 (PEP1) regulates flowering and perennial traits in a vernalization-dependent manner. Mutagenesis screens of the pep1 mutant established the role of other flowering time regulators in PEP1-parallel pathways. Here we characterized three allelic enhancers of pep1 (eop002, 085 and 091) which flower early. We mapped the causal mutations and complemented mutants with the identified gene. Using quantitative reverse transcriptase PCR and reporter lines, we determined the protein spatiotemporal expression patterns and localization within the cell. We also characterized its role in Arabidopsis thaliana using CRISPR and in A. alpina by introgressing mutant alleles into a wild-type background. These mutants carried lesions in an AAA+ ATPase of unknown function, FLOWERING REPRESSOR AAA+ ATPase 1 (AaFRAT1). AaFRAT1 was detected in the vasculature of young leaf primordia and the rib zone of flowering shoot apical meristems. At the subcellular level, AaFRAT1 was localized at the interphase between the endoplasmic reticulum and peroxisomes. Introgression lines carrying Aafrat1 alleles required less vernalization to flower and reduced number of vegetative axillary branches. By contrast, A. thaliana CRISPR lines showed weak flowering phenotypes. AaFRAT1 contributes to flowering time regulation and the perennial growth habit of A. alpina.

Auxin boosts energy generation pathways to fuel pollen maturation in barley.

Pollen grains become increasingly independent of the mother plant as they reach maturity through poorly understood developmental programs. We report that the hormone auxin is essential during barley pollen maturation to boost the expression of genes encoding almost every step of heterotrophic energy production pathways. Accordingly, auxin is necessary for the flux of sucrose and hexoses into glycolysis and to increase the levels of pyruvate and two tricarboxylic (TCA) cycle metabolites (citrate and succinate). Moreover, bioactive auxin is synthesized by the pollen-localized enzyme HvYUCCA4, supporting that pollen grains autonomously produce auxin to stimulate a specific cellular output, energy generation, that fuels maturation processes such as starch accumulation. Our results demonstrate that auxin can shift central carbon metabolism to drive plant cell development, which suggests a direct mechanism for auxin's ability to promote growth and differentiation.

The Ustilago hordei-Barley Interaction Is a Versatile System for Characterization of Fungal Effectors.

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei-barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

Beyond flowering time: diverse roles of an APETALA2-like transcription factor in shoot architecture and perennial traits.

Polycarpic perennials maintain vegetative growth after flowering. PERPETUAL FLOWERING 1 (PEP1), the orthologue of FLOWERING LOCUS C (FLC) in Arabis alpina regulates flowering and contributes to polycarpy in a vernalisation-dependent pathway. pep1 mutants do not require vernalisation to flower and have reduced return to vegetative growth as all of their axillary branches become reproductive. To identify additional genes that regulate flowering and contribute to perennial traits we performed an enhancer screen of pep1. Using mapping-by-sequencing, we cloned a mutant (enhancer of pep1-055, eop055), performed transcriptome analysis and physiologically characterised the role it plays on perennial traits in an introgression line carrying the eop055 mutation and a functional PEP1 wild-type allele. eop055 flowers earlier than pep1 and carries a lesion in the A. alpina orthologue of the APETALA2 (AP2)-like gene, TARGET OF EAT2 (AaTOE2). AaTOE2 is a floral repressor and acts upstream of SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 5 (AaSPL5). In the wild-type background, which requires cold treatment to flower, AaTOE2 regulates the age-dependent response to vernalisation. In addition, AaTOE2 ensures the maintenance of vegetative growth by delaying axillary meristem initiation and repressing flowering of axillary buds before and during cold exposure. We conclude that AaTOE2 is instrumental in fine tuning different developmental traits in the perennial life cycle of A. alpina.

Vernalization shapes shoot architecture and ensures the maintenance of dormant buds in the perennial Arabis alpina.

Perennials have a complex shoot architecture with axillary meristems organized in zones of differential bud activity and fate. This includes zones of buds maintained dormant for multiple seasons and used as reservoirs for potential growth in case of damage. The shoot of Arabis alpina, a perennial relative of Arabidopsis thaliana, consists of a zone of dormant buds placed between subapical vegetative and basal flowering branches. This shoot architecture is shaped after exposure to prolonged cold, required for flowering. To understand how vernalization ensures the maintenance of dormant buds, we performed physiological and transcriptome studies, followed the spatiotemporal changes of auxin, and generated transgenic plants. Our results demonstrate that the complex shoot architecture in A. alpina is shaped by its flowering behavior, specifically the initiation of inflorescences during cold treatment and rapid flowering after subsequent exposure to growth-promoting conditions. Dormant buds are already formed before cold treatment. However, dormancy in these buds is enhanced during, and stably maintained after, vernalization by a BRC1-dependent mechanism. Post-vernalization, stable maintenance of dormant buds is correlated with increased auxin response, transport, and endogenous indole-3-acetic acid levels in the stem. Here, we provide a functional link between flowering and the maintenance of dormant buds in perennials.

Seed coat development in explosively dispersed seeds of Cardamine hirsuta.

BACKGROUND AND AIMS: Seeds are dispersed by explosive coiling of the fruit valves in Cardamine hirsuta. This rapid coiling launches the small seeds on ballistic trajectories to spread over a 2 m radius around the parent plant. The seed surface interacts with both the coiling fruit valve during launch and subsequently with the air during flight. We aim to identify features of the seed surface that may contribute to these interactions by characterizing seed coat differentiation. METHODS: Differentiation of the outermost seed coat layers from the outer integuments of the ovule involves dramatic cellular changes that we characterize in detail at the light and electron microscopical level including immunofluorescence and immunogold labelling. KEY RESULTS: We found that the two outer integument (oi) layers of the seed coat contributed differently to the topography of the seed surface in the explosively dispersed seeds of C. hirsuta vs. the related species Arabidopsis thaliana where seed dispersal is non-explosive. The surface of A. thaliana seeds is shaped by the columella and the anticlinal cell walls of the epidermal oi2 layer. In contrast, the surface of C. hirsuta seeds is shaped by a network of prominent ridges formed by the anticlinal walls of asymmetrically thickened cells of the sub-epidermal oi1 layer, especially at the seed margin. Both the oi2 and oi1 cell layers in C. hirsuta seeds are characterized by specialized, pectin-rich cell walls that are deposited asymmetrically in the cell. CONCLUSIONS: The two outermost seed coat layers in C. hirsuta have distinct properties: the sub-epidermal oi1 layer determines the topography of the seed surface, while the epidermal oi2 layer accumulates mucilage. These properties are influenced by polar deposition of distinct pectin polysaccharides in the cell wall. Although the ridged seed surface formed by oi1 cell walls is associated with ballistic dispersal in C. hirsuta, it is not restricted to explosively dispersed seeds in the Brassicaceae.

FGB1 and WSC3 are in planta-induced ß-glucan-binding fungal lectins with different functions.

In the root endophyte Serendipita indica, several lectin-like members of the expanded multigene family of WSC proteins are transcriptionally induced in planta and are potentially involved in ß-glucan remodeling at the fungal cell wall. Using biochemical and cytological approaches we show that one of these lectins, SiWSC3 with three WSC domains, is an integral fungal cell wall component that binds to long-chain ß1-3-glucan but has no affinity for shorter ß1-3- or ß1-6-linked glucose oligomers. Comparative analysis with the previously identified ß-glucan-binding lectin SiFGB1 demonstrated that whereas SiWSC3 does not require ß1-6-linked glucose for efficient binding to branched ß1-3-glucan, SiFGB1 does. In contrast to SiFGB1, the multivalent SiWSC3 lectin can efficiently agglutinate fungal cells and is additionally induced during fungus-fungus confrontation, suggesting different functions for these two ß-glucan-binding lectins. Our results highlight the importance of the ß-glucan cell wall component in plant-fungus interactions and the potential of ß-glucan-binding lectins as specific detection tools for fungi in vivo.