Background: The development of an efficient tissue culture system is essential for advancing genetic transformation and genome editing in commercially important pineapple cultivars. However, a robust tissue culture workflow for the elite pineapple cultivar 73–50, enabling reliable transformation and plant regeneration is not established. Methods: A comparative analysis of hormone combinations, including 6-benzylaminopurine (BAP), α-naphthaleneacetic acid (NAA), picloram, and abscisic acid (ABA) was conducted. Transformation competence of 73–50 callus was tested using the iGUS reporter gene. Results: We established that 1 mg/L picloram and 0.5 µg/L ABA was the most effective combination for inducing friable embryogenic callus (FEC). FEC, composed of small, loosely associated cell clusters, is highly suitable for transformation but prone to browning during long-term culture. We optimized the conditions to minimize browning and support prolonged maintenance using a medium supplemented with 5 mg/L NAA. Transformation efficiency was demonstrated using the iGUS reporter gene, showing that FEC can be effectively transformed via both biolistic and Agrobacterium-mediated methods. For shoot regeneration, the optimal medium was found to contain 2 mg/L BAP. To standardize the assessment of callus development, we introduce a classification system describing distinct developmental stages. Conclusions: A detailed step-by-step protocol optimized for 73–50 cultivar facilitates efficient genetic improvement in pineapple, supporting both conventional transformation and DNA-free genome editing approaches.

The broad-spectrum herbicide L-phosphinothricin (PPT) irreversibly inhibits glutamine synthetase (GS) activity. Here we present cationic amino acid transporters (CAT) as mediators of PPT accumulation and susceptibility. Comparative analysis of Arabidopsis thaliana, Marchantia polymorpha, rice, and Klebsormidium nitens reveals conserved responses to GS inhibition, with absence of efficient PPT uptake underlying resistance in M. polymorpha. Transcriptomic analysis identifies four candidate transporters in A. thaliana which, when overexpressed in M. polymorpha, confer varying levels of sensitivity corresponding with PPT accumulation. AtCAT1 and AtCAT5 confer the greatest sensitivities and are required for glutamic acid uptake and endogenous nitrogen metabolism in A. thaliana, and PPT susceptibility. Molecular dynamics simulation of AtCAT5 identifies key residues involved in PPT binding, which induce the rotational flexibility of helices H1 and H6 which form an intracellular access tunnel. These findings highlight the natural diversity underlying PPT accumulation and susceptibility, which can guide herbicide resistance management strategies.

Senescence is the last critical stage of the fruit life cycle, which directly affects the maintenance of fruit quality and post harvest lifespan. The pericarp consists of various cell types, making it challenging to elucidate their roles in fruit senescence. In this study, an expression atlas of H. undatus pericarp was constructed using scRNA-seq profiles, encompassing data from thousands of individual cells. Five different algorithms, including deconvolution (SingleR and SciBet), supervised learning (RCTD and CARD), and multi-modal cross-analysis (MIA), were employed to assign 17 transcriptionally distinct cell clusters identified in single cells to four different cell types: exocarp, mesocarp, endocarp, and vascular bundles. Pseudotime trajectory analysis revealed a clear spatiotemporal transition from ROS-induced stress and disease resistance to senescence during the fruit ripening process, moving from the exocarp to the mesocarp. Previously unknown early response genes related to senescence and resistance were identified, providing novel tools for fruit senescence prediction based on mesocarp and enhancement of fruit resistance based on exocarp.

Programmed cell death (PCD) is a critical process in plant immunity, enabling the targeted elimination of infected cells to prevent the spread of pathogens. The tight regulation of PCD within plant cells is well-documented; however, specific mechanisms remain elusive or controversial. Heterotrimeric G proteins are multifunctional signaling elements consisting of three distinct subunits, Gα, Gβ, and Gγ. In Arabidopsis, the Gβγ dimer serves as a positive regulator of plant defense. Conversely, in species such as rice, maize, cotton, and tomato, mutants deficient in Gβ exhibit constitutively active defense responses, suggesting a contrasting negative role for Gβ in defense mechanisms within these plants. Using a transient overexpression approach in addition to knockout mutants, we observed that Gβγ enhanced cell death progression and elevated the accumulation of reactive oxygen species in a similar manner across Arabidopsis, tomato, and Nicotiana benthamiana, suggesting a conserved G protein role in PCD regulation among diverse plant species. The enhancement of PCD progression was cooperatively regulated by Gβγ and one Gα, XLG2. We hypothesize that G proteins participate in two distinct mechanisms regulating the initiation and progression of PCD in plants. We speculate that G proteins may act as guardees, the absence of which triggers PCD. However, in Arabidopsis, this G protein guarding mechanism appears to have been lost in the course of evolution.

Abstract The immune system is crucial for plant survival, although its activation diverts resources away from other important processes, such as growth and reproduction. To balance the need for an effective immune response with the need to maintain overall fitness, plants have evolved immune priming. The priming or pre-activation of defence mechanisms allows plant cells to respond faster and more efficiently to an incoming threat without triggering the actual defence response. In this study, we reveal a novel molecular mechanism of temperature dependent immune priming mediated by the Arabidopsis extra-large GTP binding protein 2 (XLG2) contributing to resistance against pathogens such as Fusarium oxysporum and Pseudomonas syringae . We found that XLG2 rapidly accumulates in the nucleus upon temperature elevation and that nuclear localization is essential for XLG2-mediated defence responses. XLG2 directly interacts with several defence-related transcription factors and stabilizes transcription activation complexes, such as nonexpressor of pathogenesis related genes 1 (NPR1) and TGACG sequence-specific binding protein (TGA2). Our observations suggest that XLG2 integrates abiotic and biotic stress signals providing an important balance between immunity and fitness and thus enabling plants to adequately respond to a changing environment.

Heterotrimeric GTP-binding proteins (G proteins), consisting of Gα, Gβ and Gγ subunits, transduce signals from a diverse range of extracellular stimuli, resulting in the regulation of numerous cellular and physiological functions in Eukaryotes. According to the classic G protein paradigm established in animal models, the bound guanine nucleotide on a Gα subunit, either guanosine diphosphate (GDP) or guanosine triphosphate (GTP) determines the inactive or active mode, respectively. In plants, there are two types of Gα subunits: canonical Gα subunits structurally similar to their animal counterparts and unconventional extra-large Gα subunits (XLGs) containing a C-terminal domain homologous to the canonical Gα along with an extended N-terminal domain. Both Gα and XLG subunits interact with Gβγ dimers and regulator of G protein signalling (RGS) protein. Plant G proteins are implicated directly or indirectly in developmental processes, stress responses, and innate immunity. It is established that despite the substantial overall similarity between plant and animal Gα subunits, they convey signalling differently including the mechanism by which they are activated. This review emphasizes the unique characteristics of plant Gα subunits and speculates on their unique signalling mechanisms.

AbstractHeterotrimeric G protein Gβ‐deficient mutants in rice and maize display constitutive immune responses, whereas Arabidopsis Gβ mutants show impaired defense, suggesting the existence of functional differences between monocots and dicots. Using CRISPR/Cas9, we produced one hemizygous tomato line with a mutated SlGB1 Gβ gene. Homozygous slgb1 knockout mutants exhibit all the hallmarks of autoimmune mutants, including development of necrotic lesions, constitutive expression of defense‐related genes, and high endogenous levels of salicylic acid (SA) and reactive oxygen species, resulting in early seedling lethality. Virus‐induced silencing of Gβ in cotton reproduced the symptoms observed in tomato mutants, confirming that the autoimmune phenotype is not limited to monocot species but is also shared by dicots. Even though multiple genes involved in SA and ethylene signaling are highly induced by Gβ silencing in tomato and cotton, co‐silencing of SA or ethylene signaling components in cotton failed to suppress the lethal phenotype, whereas co‐silencing of the oxidative burst oxidase RbohD can repress lethality. Despite the autoimmune response observed in slgb1 mutants, we show that SlGB1 is a positive regulator of the pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) response in tomato. We speculate that the phenotypic differences observed between Arabidopsis and tomato/cotton/rice/maize Gβ knockouts do not necessarily reflect divergences in G protein‐mediated defense mechanisms.

The extra-large guanosine-5'-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

In animals, endocytosis of a seven-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic G protein-mediated signaling, depending on the bias of the receptor or ligand, which determines how much one transduction pathway is used compared to another. In Arabidopsis thaliana, GPCRs are not required for G protein-coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the seven-transmembrane protein AtRGS1 modulates G protein signaling through ligand-dependent endocytosis, which initiates derepression of signaling without the involvement of canonical arrestins. Here, we found that endocytosis of AtRGS1 initiated from two separate pools of plasma membrane: sterol-dependent domains and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and candidate arrestin-like adaptors. Ligand identity (either the pathogen-associated molecular pattern flg22 or the sugar glucose) determined the origin of AtRGS1 endocytosis. Different trafficking origins and trajectories led to different cellular outcomes. Thus, in this system, compartmentation with its associated signalosome architecture drives biased signaling.

Variation in grain size, a major determinant of grain yield and quality in cereal crops, is determined by both the plant's genetic potential and the available assimilate to fill the grain in the absence of stress. This study investigated grain size variation in response to variation in assimilate supply in sorghum using a diversity panel (n = 837) and a backcross-nested association mapping population (n = 1421) across four experiments. To explore the effects of genetic potential and assimilate availability on grain size, the top half of selected panicles was removed at anthesis. Results showed substantial variation in five grain size parameters with high heritability. Artificial reduction in grain number resulted in a general increase in grain weight, with the extent of the increase varying across genotypes. Genome-wide association studies identified 44 grain size quantitative trait locus (QTL) that were likely to act on assimilate availability and 50 QTL that were likely to act on genetic potential. This finding was further supported by functional enrichment analysis and co-location analysis with known grain number QTL and candidate genes. RNA interference and overexpression experiments were conducted to validate the function of one of the identified gene, SbDEP1, showing that SbDEP1 positively regulates grain number and negatively regulates grain size by controlling primary branching in sorghum. Haplotype analysis of SbDEP1 suggested a possible role in racial differentiation. The enhanced understanding of grain size variation in relation to assimilate availability presented in this study will benefit sorghum improvement and have implications for other cereal crops.

Biased signaling occurs when different ligands that are directed at the same receptor launch different cellular outcomes. Because of their pharmacological importance, we know the most about biased ligands and little is known about other mechanisms to achieve signaling bias. In the canonical animal G protein system, endocytosis of a 7-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic signaling depending on the bias. In Arabidopsis, GPCRs are not required for G protein coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the prototype 7-transmembrane Regulator of G Signaling 1 protein AtRGS1 modulates G signaling and through ligand-dependent endocytosis, de-repression of signaling is initiated but canonical arrestins are not involved. Endocytosis initiates from two separate pools of plasma membrane: sterol-dependent domains, possibly lipid rafts, and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and newly-discovered arrestin-like adaptors. Different trafficking origins and trajectories lead to different cellular outcomes. Thus, compartmentation with its attendant signalosome architecture is a previously unknown mechanism to drive biased signaling.

G protein α subunits in Arabidopsis can function independently of guanine nucleotide exchange.

G-proteins are universal signal transducers mediating many cellular responses. Plant G-protein signaling has been modeled on the well-established animal paradigm but accumulated experimental evidence indicates that G-protein-dependent signaling in plants has taken a very different evolutionary path. Here we review the differences between plant and animal G-proteins reported over past two decades. Most importantly, while in animal systems the G-protein signaling cycle is activated by seven transmembrane-spanning G-protein coupled receptors, the existence of these type of receptors in plants is highly controversial. Instead plant G-proteins have been proven to be functionally associated with atypical receptors such as the Arabidopsis RGS1 and a number of receptor-like kinases. We propose that, instead of the GTP/GDP cycle used in animals, plant G-proteins are activated/de-activated by phosphorylation/de-phosphorylation. We discuss the need of a fresh new look at these signaling molecules and provide a hypothetical model that departs fromthe accepted animal paradigm.

Studying the natural defense mechanisms developed by model plants such as Arabidopsis is an important approach towards the improvement of crop species. The availability of mutants as well as the relative easiness to silence any gene in Arabidopsis provides an invaluable source of genotypes that can be used to discover new elements involved in the defense response. Here we describe simple and reliable methods to evaluate susceptibility/resistance to the pathogenic fungus Alternaria brassicicola and the viral pathogen Turnip mosaic virus.

Rapid diversification of the XLG family of Gα proteins may have enabled plants to adapt to the variable land environment.

Heterotrimeric G-proteins, consisting of Gα, Gβ and Gγ subunits, are important signal transducers in eukaryotes. In plants, G-protein-mediated signaling contributes to defense against a range of fungal and bacterial pathogens. Here we studied response of G-protein-deficient mutants to ssRNA viruses representing 2 different families: Cucumber mosaic virus (CMV) (Bromoviridae) and Turnip mosaic virus (TuMV) (Potyviridae). We found that development of spreading necrosis on infected plants was suppressed in the Gβ-deficient mutant (agb1-2) compared to wild type and Gα-deficient mutant (gpa1-4). In accordance, ion leakage caused by viral infection was also significantly reduced in agb1-2 compared to wild type and gpa1-4. Nevertheless, both viruses replicated better in agb1-2 plants, while gpa1-4 was similar to wild type. Analysis of pathogenesis-related genes showed that Gβ negatively regulated salicylic acid, jasmonic acid and abscisic acid marker genes during CMV and TuMV infections. Interestingly, analysis of salicylic acid deficient transgenic plants indicated that salicylic acid did not affect resistance against these viruses and did not influence the Gβ-mediated defense response. We conclude that heterotrimeric G-proteins play a positive role in defense against viral pathogens probably by promoting cell death.

Protein-protein interaction studies provide useful insights into biological processes taking place within the living cell. A number of techniques are available to unravel large structural protein complexes, functional protein modules, and temporary protein associations occurring during signal transduction. The choice of method depends on the nature of the proteins and the interaction being studied. Here we present an optimized and simplified yeast three-hybrid method for analysis of protein interactions involving three components.

In animals, heterotrimeric G proteins, comprising G alpha, G beta, and G gamma subunits, are molecular switches whose function tightly depends on G alpha and G beta gamma interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve G beta gamma, but not G alpha. We report here that the G beta gamma dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, G beta, and G gamma are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and G beta gamma functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis G beta-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible G beta gamma dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant G alpha subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with G beta gamma dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical G alpha subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.