Ethylene controls three-dimensional growth involving reduced auxin levels in the moss Physcomitrium patens.

The conquest of land by plants was concomitant with, and possibly enabled by, the evolution of three-dimensional (3D) growth. The moss Physcomitrium patens provides a model system for elucidating molecular mechanisms in the initiation of 3D growth. Here, we investigate whether the phytohormone ethylene, which is believed to have been a signal before land plant emergence, plays a role in 3D growth regulation in P. patens. We report ethylene controls 3D gametophore formation, based on results from exogenously applied ethylene and genetic manipulation of PpEIN2, which is a central component in the ethylene signaling pathway. Overexpression (OE) of PpEIN2 activates ethylene responses and leads to earlier formation of gametophores with fewer gametophores produced thereafter, phenocopying ethylene-treated wild-type. Conversely, Ppein2 knockout mutants, which are ethylene insensitive, show initially delayed gametophore formation with more gametophores produced later. Furthermore, pharmacological and biochemical analyses reveal auxin levels are decreased in the OE lines but increased in the knockout mutants. Our results suggest that evolutionarily, ethylene and auxin molecular networks were recruited to build the plant body plan in ancestral land plants. This might have played a role in enabling ancient plants to acclimate to the continental surfaces of the planet.

The plant hormone ethylene promotes abiotic stress tolerance in the liverwort Marchantia polymorpha.

Plants are often faced with an array of adverse environmental conditions and must respond appropriately to grow and develop. In angiosperms, the plant hormone ethylene is known to play a protective role in responses to abiotic stress. Here we investigated whether ethylene mediates resistance to abiotic stress in the liverwort Marchantia polymorpha, one of the most distant land plant relatives of angiosperms. Using existing M. polymorpha knockout mutants of Mpein3, and Mpctr1, two genes in the ethylene signaling pathway, we examined responses to heat, salinity, nutrient deficiency, and continuous far-red light. The Mpein3 and Mpctr1 mutants were previously shown to confer ethylene insensitivity and constitutive ethylene responses, respectively. Using mild or sub-lethal doses of each stress treatment, we found that Mpctr1 mutants displayed stress resilience similar to or greater than the wild type. In contrast, Mpein3 mutants showed less resilience than the wild type. Consistent with ethylene being a stress hormone, we demonstrated that ethylene production is enhanced by each stress treatment. These results suggest that ethylene plays a role in protecting against abiotic stress in M. polymorpha, and that ethylene has likely been conserved as a stress hormone since before the evolutionary divergence of bryophytes from the land plant lineage approximately 450 Ma.

A transporter of 1-aminocyclopropane-1-carboxylic acid affects thallus growth and fertility in Marchantia polymorpha.

In seed plants, 1-aminocyclopropane-1-carboxylic acid (ACC) is the precursor of the plant hormone ethylene but also has ethylene-independent signaling roles. Nonseed plants produce ACC but do not efficiently convert it to ethylene. In Arabidopsis thaliana, ACC is transported by amino acid transporters, LYSINE HISTIDINE TRANSPORTER 1 (LHT1) and LHT2. In nonseed plants, LHT homologs have been uncharacterized. Here, we isolated an ACC-insensitive mutant (Mpain) that is defective in ACC uptake in the liverwort Marchantia polymorpha. Mpain contained a frameshift mutation (1 bp deletion) in the MpLHT1 coding sequence, and was complemented by expression of a wild-type MpLHT1 transgene. Additionally, ACC insensitivity was re-created in CRISPR/Cas9-Mplht1 knockout mutants. We found that MpLHT1 can also transport l-hydroxyproline and l-histidine. We examined the physiological functions of MpLHT1 in vegetative growth and reproduction based on mutant phenotypes. Mpain and Mplht1 plants were smaller and developed fewer gemmae cups compared to wild-type plants. Mplht1 mutants also had reduced fertility, and archegoniophores displayed early senescence. These findings reveal that MpLHT1 serves as an ACC and amino acid transporter in M. polymorpha and has diverse physiological functions. We propose that MpLHT1 contributes to homeostasis of ACC and other amino acids in M. polymorpha growth and reproduction.

ConducTORs of a Signaling Symphony: Metabolic and Hormone Responses Converge on TOR and EIN2 in plants.

Development is coordinated by dozens of signals that act in overlapping pathways to orchestrate multicellular growth. Understanding how signaling pathways intersect and diverge at a molecular level is critical to predicting how organisms will react to dynamic environmental conditions. In plants, two antagonistic signaling hubs are strictly required to sense and respond to many nutrients and hormones: TARGET OF RAPAMYCIN (TOR) and ETHYLENE INSENSITIVE 2 (EIN2). In this Landmark report, Fu et al. discover that TOR and EIN2 directly interact to choreograph growth and define an unexpected molecular mechanism at the intersection of hormonal and metabolic signaling networks1.

Something old, something new: Conservation of the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid as a signaling molecule.

In seed plants, 1-amino-cyclopropane-1-carboxylic acid (ACC) is the well-known precursor of the plant hormone ethylene. In nonseed plants, the current view is that ACC is produced but is inefficiently converted to ethylene. Distinct responses to ACC that are uncoupled from ethylene biosynthesis have been discovered in diverse aspects of growth and development in liverworts and angiosperms, indicating that ACC itself can function as a signal. Evolutionarily, ACC may have served as a signal before acquiring its role as the ethylene precursor in seed plants. These findings pave the way for unraveling a potentially conserved ACC signaling pathway in plants and have ramifications for the use of ACC as a substitute for ethylene treatment in seed plants.

1-Aminocyclopropane-1-carboxylic acid stimulates tomato pollen tube growth independently of ethylene receptors.

The plant hormone ethylene plays vital roles in plant development, including pollen tube (PT) growth. Many studies have used the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as a tool to trigger ethylene signaling. Several studies have suggested that ACC can act as a signal molecule independently of ethylene, inducing responses that are distinct from those induced by ethylene. In this study, we confirmed that ethylene receptor function is essential for promoting PT growth in tomato, but interestingly, we discovered that ACC itself can act as a signal that also promotes PT growth. Exogenous ACC stimulated PT growth even when ethylene perception was inhibited either chemically by treating with 1-methylcyclopropene (1-MCP) or genetically by using the ethylene-insensitive Never Ripe (NR) mutant. Treatment with aminoethoxyvinylglycine, which reduces endogenous ACC levels, led to a reduction of PT growth, even in the NR mutants. Furthermore, GUS activity driven by an EIN3 Binding Site promoter (EBS:GUS transgene) was triggered by ACC in the presence of 1-MCP. Taken together, these results suggest that ACC signaling can bypass the ethylene receptor step to stimulate PT growth and EBS driven gene expression.

Ethylene-independent functions of the ethylene precursor ACC in Marchantia polymorpha.

The plant hormone ethylene has many roles in growth and development1. In seed plants, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is converted into ethylene by ACC oxidase (ACO), and treatment with ACC induces ethylene responses2. However, non-seed plants lack ACO homologues3-8, which led us to examine the relationship between ACC and ethylene in the liverwort Marchantia polymorpha. Here, we demonstrate that ACC and ethylene can induce divergent growth responses in Marchantia. Ethylene increases plant and gemma size, induces more gemma cups and promotes gemmae dormancy. As predicted, Mpctr1-knockout mutants display constitutive ethylene responses, whereas Mpein3-knockout mutants exhibit ethylene insensitivity. Compared with the wild type, Mpctr1 gemmae have more and larger epidermal cells, whereas Mpein3 gemmae have fewer and smaller epidermal cells, suggesting that ethylene promotes cell division and growth in developing gemmae. By contrast, ACC treatment inhibits gemma growth and development by suppressing cell division, even in the Mpein3-knockout alleles. Knockout mutants of one or both ACC SYNTHASE (ACS) gene homologues produce negligible levels of ACC, have more and larger gemma cups, and have more-expanded thallus branches. Mpacs2 and Mpacs1 Mpacs2 gemmae also display a high frequency of abnormal apical notches (meristems) that are not observed in ethylene mutants. These findings reveal that ethylene and ACC have distinct functions, and suggest that ACC is a signalling molecule in Marchantia. ACC may be an evolutionarily conserved signal that predates its efficient conversion to ethylene in higher plants.

Ethylene-independent signaling by the ethylene precursor ACC in Arabidopsis ovular pollen tube attraction.

The phytohormone ethylene has numerous effects on plant growth and development. Its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is a non-proteinogenic amino acid produced by ACC SYNTHASE (ACS). ACC is often used to induce ethylene responses. Here, we demonstrate that ACC exhibits ethylene-independent signaling in Arabidopsis thaliana reproduction. By analyzing an acs octuple mutant with reduced seed set, we find that ACC signaling in ovular sporophytic tissue is involved in pollen tube attraction, and promotes secretion of the pollen tube chemoattractant LURE1.2. ACC activates Ca2+-containing ion currents via GLUTAMATE RECEPTOR-LIKE (GLR) channels in root protoplasts. In COS-7 cells expressing moss PpGLR1, ACC induces the highest cytosolic Ca2+ elevation compared to all twenty proteinogenic amino acids. In ovules, ACC stimulates transient Ca2+ elevation, and Ca2+ influx in octuple mutant ovules rescues LURE1.2 secretion. These findings uncover a novel ACC function and provide insights for unraveling new physiological implications of ACC in plants.

The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

The root-knot nematode Meloidogyne incognita produces a functional mimic of the Arabidopsis INFLORESCENCE DEFICIENT IN ABSCISSION signaling peptide.

INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is a signaling peptide that regulates cell separation in Arabidopsis including floral organ abscission and lateral root emergence. IDA is highly conserved in dicotyledonous flowering plant genomes. IDA-like sequences were also found in the genomic sequences of root-knot nematodes, Meloidogyne spp., which are globally deleterious pathogens of agriculturally important plants, but the role of these genes is unknown. Exogenous treatment of the Arabidopsis ida mutant with synthetic peptide identical to the M. incognita IDA-like 1 (MiIDL1) protein sequence minus its N-terminal signal peptide recovered both the abscission and root architecture defects. Constitutive expression of the full-length MiIDL1 open reading frame in the ida mutant substantially recovered the delayed floral organ abscission phenotype whereas transformants expressing a construct missing the MiIDL1 signal peptide retained the delayed abscission phenotype. Importantly, wild-type Arabidopsis plants harboring an MiIDL1-RNAi construct and infected with nematodes had approximately 40% fewer galls per root than control plants. Thus, the MiIDL1 gene produces a functional IDA mimic that appears to play a role in successful gall development on Arabidopsis roots.

Establishment of Dimethyl Labeling-based Quantitative Acetylproteomics in Arabidopsis.

Protein acetylation, one of many types of post-translational modifications (PTMs), is involved in a variety of biological and cellular processes. In the present study, we applied both CsCl density gradient (CDG) centrifugation-based protein fractionation and a dimethyl-labeling-based 4C quantitative PTM proteomics workflow in the study of dynamic acetylproteomic changes in Arabidopsis. This workflow integrates the dimethyl chemical labeling with chromatography-based acetylpeptide separation and enrichment followed by mass spectrometry (MS) analysis, the extracted ion chromatogram (XIC) quantitation-based computational analysis of mass spectrometry data to measure dynamic changes of acetylpeptide level using an in-house software program, named Stable isotope-based Quantitation-Dimethyl labeling (SQUA-D), and finally the confirmation of ethylene hormone-regulated acetylation using immunoblot analysis. Eventually, using this proteomic approach, 7456 unambiguous acetylation sites were found from 2638 different acetylproteins, and 5250 acetylation sites, including 5233 sites on lysine side chain and 17 sites on protein N termini, were identified repetitively. Out of these repetitively discovered acetylation sites, 4228 sites on lysine side chain (i.e. 80.5%) are novel. These acetylproteins are exemplified by the histone superfamily, ribosomal and heat shock proteins, and proteins related to stress/stimulus responses and energy metabolism. The novel acetylproteins enriched by the CDG centrifugation fractionation contain many cellular trafficking proteins, membrane-bound receptors, and receptor-like kinases, which are mostly involved in brassinosteroid, light, gravity, and development signaling. In addition, we identified 12 highly conserved acetylation site motifs within histones, P-glycoproteins, actin depolymerizing factors, ATPases, transcription factors, and receptor-like kinases. Using SQUA-D software, we have quantified 33 ethylene hormone-enhanced and 31 hormone-suppressed acetylpeptide groups or called unique PTM peptide arrays (UPAs) that share the identical unique PTM site pattern (UPSP). This CDG centrifugation protein fractionation in combination with dimethyl labeling-based quantitative PTM proteomics, and SQUA-D may be applied in the quantitation of any PTM proteins in any model eukaryotes and agricultural crops as well as tissue samples of animals and human beings.

Land Plant Model Systems Branch Out.

The genome of the liverwort Marchantia polymorpha is an important step toward development of a new plant model system (Bowman et al., 2017). Liverworts may be the sister taxon to all other land plants, and the genome shows features that illuminate the ancestor of all land plants and give insights into how plant systems function and evolved.

Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor.

The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane-bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor-interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi-fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1-1 and etr1-2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis.

Molecular association of Arabidopsis RTH with its homolog RTE1 in regulating ethylene signaling.

The plant hormone ethylene affects many biological processes during plant growth and development. Ethylene is perceived by ethylene receptors at the endoplasmic reticulum (ER) membrane. The ETR1 ethylene receptor is positively regulated by the transmembrane protein RTE1, which localizes to the ER and Golgi apparatus. The RTE1 gene family is conserved in animals, plants, and lower eukaryotes. In Arabidopsis, RTE1-HOMOLOG (RTH) is the only homolog of the Arabidopsis RTE1 gene family. The regulatory function of the Arabidopsis RTH in ethylene signaling and plant growth is largely unknown. The present study shows Arabidopsis RTH gene expression patterns, protein co-localization with the ER and Golgi apparatus, and the altered ethylene response phenotype when RTH is knocked out or overexpressed in Arabidopsis. Compared with rte1 mutants, rth mutants exhibit less sensitivity to exogenous ethylene, while RTH overexpression confers ethylene hypersensitivity. Genetic analyses indicate that Arabidopsis RTH might not directly regulate the ethylene receptors. RTH can physically interact with RTE1, and evidence supports that RTH might act via RTE1 in regulating ethylene responses and signaling. The present study advances our understanding of the regulatory function of the Arabidopsis RTE1 gene family members in ethylene signaling.

Field Guide to Plant Model Systems.

For the past several decades, advances in plant development, physiology, cell biology, and genetics have relied heavily on the model (or reference) plant Arabidopsis thaliana. Arabidopsis resembles other plants, including crop plants, in many but by no means all respects. Study of Arabidopsis alone provides little information on the evolutionary history of plants, evolutionary differences between species, plants that survive in different environments, or plants that access nutrients and photosynthesize differently. Empowered by the availability of large-scale sequencing and new technologies for investigating gene function, many new plant models are being proposed and studied.

Transcriptome Profiling of the Green Alga Spirogyra pratensis (Charophyta) Suggests an Ancestral Role for Ethylene in Cell Wall Metabolism, Photosynthesis, and Abiotic Stress Responses.

It is well known that ethylene regulates a diverse set of developmental and stress-related processes in angiosperms, yet its roles in early-diverging embryophytes and algae are poorly understood. Recently, it was shown that ethylene functions as a hormone in the charophyte green alga Spirogyra pratensis Since land plants evolved from charophytes, this implies conservation of ethylene as a hormone in green plants for at least 450 million years. However, the physiological role of ethylene in charophyte algae has remained unknown. To gain insight into ethylene responses in Spirogyra, we used mRNA sequencing to measure changes in gene expression over time in Spirogyra filaments in response to an ethylene treatment. Our analyses show that at the transcriptional level, ethylene predominantly regulates three processes in Spirogyra: (1) modification of the cell wall matrix by expansins and xyloglucan endotransglucosylases/hydrolases, (2) down-regulation of chlorophyll biosynthesis and photosynthesis, and (3) activation of abiotic stress responses. We confirmed that the photosynthetic capacity and chlorophyll content were reduced by an ethylene treatment and that several abiotic stress conditions could stimulate cell elongation in an ethylene-dependent manner. We also found that the Spirogyra transcriptome harbors only 10 ethylene-responsive transcription factor (ERF) homologs, several of which are regulated by ethylene. These results provide an initial understanding of the hormonal responses induced by ethylene in Spirogyra and help to reconstruct the role of ethylene in ancestral charophytes prior to the origin of land plants.

Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.