Small but powerful: RALF peptides in plant adaptive and developmental responses.

Plants live in a highly dynamic environment and require to rapidly respond to a plethora of environmental stimuli, so that to maintain their optimal growth and development. A small plant peptide, rapid alkalization factor (RALF), can rapidly increase the pH value of the extracellular matrix in plant cells. RALFs always function with its corresponding receptors. Mechanistically, effective amount of RALF is induced and released at the critical period of plant growth and development or under different external environmental factors. Recent studies also highlighted the role of RALF peptides as important regulators in plant intercellular communications, as well as their operation in signal perception and as ligands for different receptor kinases on the surface of the plasma membrane, to integrate various environmental cues. In this context, understanding the fine-print of above processes may be essential to solve the problems of crop adaptation to various harsh environments under current climate trends scenarios, by genetic means. This paper summarizes the current knowledge about the structure and diversity of RALF peptides and their roles in plant development and response to stresses, highlighting unanswered questions and problems to be solved.

Predisposing deleterious variants in the cancer-associated human kinases in the global populations.

Human kinases play essential and diverse roles in the cellular activities of maintaining homeostasis and growth. Genetic mutations in the genes encoding the kinases (or phosphotransferases) have been linked with various types of cancers. In this study, we cataloged mutations in 500 kinases genes in >65,000 individuals of global populations from the Human Genetic Diversity Project (HGDP) and ExAC databases, and assessed their potentially deleterious impact by using the in silico tools SIFT, Polyphen2, and CADD. The analysis highlighted 35 deleterious non-synonymous SNVs in the ExAC and 5 SNVs in the HGDP project. Notably, a higher number of deleterious mutations was observed in the Non-Finnish Europeans (26 SNVs), followed by the Africans (14 SNVs), East Asians (13 SNVs), and South Asians (12 SNVs). The gene set enrichment analysis highlighted NTRK1 and FGFR3 being most significantly enriched among the kinases. The gene expression analysis revealed over-expression of NTRK1 in liver cancer, whereas, FGFR3 was found over-expressed in lung, breast, and liver cancers compared to their expression in the respective normal tissues. Also, 13 potential drugs were identified that target the NTRK1 protein, whereas 6 potential drugs for the FGFR3 target were identified. Taken together, the study provides a framework for exploring the predisposing germline mutations in kinases to suggest the underlying pathogenic mechanisms in cancers. The potential drugs are also suggested for personalized cancer management.

Redundant role of OsCNGC4 and OsCNGC5 encoding cyclic nucleotide-gated channels in rice pollen germination and tube growth.

In staple crops, such as rice (Oryza sativa L.), pollen plays a crucial role in seed production. However, the molecular mechanisms underlying rice pollen germination and tube growth remain underexplored. Notably, we recently uncovered the redundant expression and mutual interaction of two rice genes encoding cyclic nucleotide-gated channels (CNGCs), OsCNGC4 and OsCNGC5, in mature pollen. Building on these findings, the current study focused on clarifying the functional roles of these two genes in pollen germination and tube growth. To overcome functional redundancy, we produced gene-edited rice plants with mutations in both genes using the CRISPR-Cas9 system. The resulting homozygous OsCNGC4 and OsCNGC5 gene-edited mutants (oscngc4/5) exhibited significantly lower pollen germination rates than the wild type (WT), along with severely reduced fertility. Transcriptome analysis of the double oscngc4/5 mutant revealed downregulation of genes related to receptor kinases, transporters, and cell wall metabolism. To identify the direct regulators of OsCNGC4, which form a heterodimer with OsCNGC5, we screened a yeast two-hybrid library containing rice cDNAs from mature anthers. Subsequently, we identified two calmodulin isoforms (CaM1-1 and CaM1-2), NETWORKED 2 A (NET2A), and proline-rich extension-like receptor kinase 13 (PERK13) proteins as interactors of OsCNGC4, suggesting its roles in regulating Ca2+ channel activity and F-actin organization. Overall, our results suggest that OsCNGC4 and OsCNGC5 may play critical roles in pollen germination and elongation by regulating the Ca2+ gradient in growing pollen tubes.

Kinome-Wide Virtual Screening by Multi-Task Deep Learning.

Deep learning is a machine learning technique to model high-level abstractions in data by utilizing a graph composed of multiple processing layers that experience various linear and non-linear transformations. This technique has been shown to perform well for applications in drug discovery, utilizing structural features of small molecules to predict activity. Here, we report a large-scale study to predict the activity of small molecules across the human kinome-a major family of drug targets, particularly in anti-cancer agents. While small-molecule kinase inhibitors exhibit impressive clinical efficacy in several different diseases, resistance often arises through adaptive kinome reprogramming or subpopulation diversity. Polypharmacology and combination therapies offer potential therapeutic strategies for patients with resistant diseases. Their development would benefit from a more comprehensive and dense knowledge of small-molecule inhibition across the human kinome. Leveraging over 650,000 bioactivity annotations for more than 300,000 small molecules, we evaluated multiple machine learning methods to predict the small-molecule inhibition of 342 kinases across the human kinome. Our results demonstrated that multi-task deep neural networks outperformed classical single-task methods, offering the potential for conducting large-scale virtual screening, predicting activity profiles, and bridging the gaps in the available data.

FAM20A is a golgi-localized Type II transmembrane protein.

Family with sequence similarity 20, member A (FAM20A) is a pseudo-kinase in the secretory pathway and is essential for enamel formation in humans. Here we examine if FAM20A is a membrane-associated protein. We show that the full-length FAM20A can be purified from HEK293 cells transfected with a FAM20A-expresing construct. Further, it is only found in the membrane fraction, but not in the soluble fraction, of cell lysate. Consistently, it is not secreted out of the expressing cells. Moreover, it is co-localized with GM130, a cis-Golgi network marker, and membrane topology analysis indicates that it has its C-terminus oriented towards the lumen of the organelle. Our results support that FAM20A is a Type II transmembrane protein within the secretory compartments.

Post-ischemic ubiquitination at the postsynaptic density reversibly influences the activity of ischemia-relevant kinases.

Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface ( https://hochrainerlab.shinyapps.io/StrokeUbiOmics/ ). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury.

A global phosphosite-correlated network map of Thousand And One Kinase 1 (TAOK1).

Thousand and one amino acid kinase 1 (TAOK1) is a sterile 20 family Serine/Threonine kinase linked to microtubule dynamics, checkpoint signaling, DNA damage response, and neurological functions. Molecular-level alterations of TAOK1 have been associated with neurodevelopment disorders and cancers. Despite their known involvement in physiological and pathophysiological processes, and as a core member of the hippo signaling pathway, the phosphoregulatory network of TAOK1 has not been visualized. Aimed to explore this network, we first analyzed the predominantly detected and differentially regulated TAOK1 phosphosites in global phosphoproteome datasets across diverse experimental conditions. Based on 709 qualitative and 210 quantitative differential cellular phosphoproteome datasets that were systematically assembled, we identified that phosphorylation at Ser421, Ser9, Ser965, and Ser445 predominantly represented TAOK1 in almost 75% of these datasets. Surprisingly, the functional role of all these phosphosites in TAOK1 remains unexplored. Hence, we employed a robust strategy to extract the phosphosites in proteins that significantly correlated in expression with predominant TAOK1 phosphosites. This led to the first categorization of the phosphosites including those in the currently known and predicted interactors, kinases, and substrates, that positively/negatively correlated with the expression status of each predominant TAOK1 phosphosites. Subsequently, we also analyzed the phosphosites in core proteins of the hippo signaling pathway. Based on the TAOK1 phosphoregulatory network analysis, we inferred the potential role of the predominant TAOK1 phosphosites. Especially, we propose pSer9 as an autophosphorylation and TAOK1 kinase activity-associated phosphosite and pS421, the most frequently detected phosphosite in TAOK1, as a significant regulatory phosphosite involved in the maintenance of genome integrity. Considering that the impact of all phosphosites that predominantly represent each kinase is essential for the efficient interpretation of global phosphoproteome datasets, we believe that the approach undertaken in this study is suitable to be extended to other kinases for accelerated research.

Genome sequencing of drake semen micobiome with correlation with their compositions, sources and potential mechanisms affecting semen quality.

Artificial insemination (AI) technology has greatly promoted the development of the chicken industry. Recently, AI technology has also begun to be used in the duck industry, but there are some problems. Numerous researchers have shown that microbes colonizing in semen can degrade semen quality, and AI can increase the harmful microbial load in hen's reproductive tract. Different from the degraded external genitalia of roosters, drakes have well-developed external genitalia, which may cause drake semen to be more susceptible to microbial contamination. However, information on the compositions, sources, and effects of semen microbes on semen quality remains unknown in drakes. In the current study, high-throughput sequencing technology was used to detect microbial communities in drake semen, environmental swabs, cloacal swabs, and the spermaduct after quantifying the semen quality of drakes to investigate the effects of microbes in the environment, cloaca, and spermaduct on semen microbiota and the relationships between semen microbes and semen quality. Taxonomic analysis showed that the microbes in the semen, environment, cloaca, and spermaduct samples were all classified into 4 phyla and 25 genera. Firmicutes and Proteobacteria were the dominant phyla. Phyllobacterium only existed in the environment, while Marinococcus did not exist in the cloaca. Of the 24 genera present in semen: Brachybacterium, Brochothrix, Chryseobacterium, Kocuria, Marinococcus, Micrococcus, Rothia, Salinicoccus, and Staphylococcus originated from the environment; Achromobacter, Aerococcus, Corynebacterium, Desemzia, Enterococcus, Jeotgalicoccus, Pseudomonas, Psychrobacter, and Turicibacter originated from the cloaca; and Agrobacterium, Carnobacterium, Chelativorans, Devosia, Halomonas, and Oceanicaulis originated from the spermaduct. In addition, K-means clustering analysis showed that semen samples could be divided into 2 clusters based on microbial compositions, and compared with cluster 1, the counts of Chelativorans (P < 0.05), Devosia (P < 0.01), Halomonas (P < 0.05), and Oceanicaulis (P < 0.05) were higher in cluster 2, while the sperm viability (P < 0.05), total sperm number (P < 0.01), and semen quality factor (SQF) (P < 0.01) were lower in cluster 2. Furthermore, functional prediction analysis of microbes showed that the activities of starch and sucrose metabolism, phosphotransferase system, ABC transporters, microbial metabolism in diverse environments, and quorum sensing pathways between cluster 1 and cluster 2 were significantly different (P < 0.05). Overall, environmental/cloacal microbes resulted in semen contamination, and microbes from the Chelativorans, Devosia, Halomonas, and Oceanicaulis genera may have negative effects on semen quality in drakes by affecting the activities of starch and sucrose metabolism, phosphotransferase system, ABC transporters, and quorum sensing pathways that are associated with carbohydrate metabolism. These data will provide a basis for developing strategies to prevent microbial contamination of drake semen.

Phosphorylation of the alpha-I motif in SYMRK drives root nodule organogenesis.

Symbiosis receptor-like kinase SYMRK is required for root nodule symbiosis between legume plants and nitrogen-fixing bacteria. To understand symbiotic signaling from SYMRK, we determined the crystal structure to 1.95 Å and mapped the phosphorylation sites onto the intracellular domain. We identified four serine residues in a conserved "alpha-I" motif, located on the border between the kinase core domain and the flexible C-terminal tail, that, when phosphorylated, drives organogenesis. Substituting the four serines with alanines abolished symbiotic signaling, while substituting them with phosphorylation-mimicking aspartates induced the formation of spontaneous nodules in the absence of bacteria. These findings show that the signaling pathway controlling root nodule organogenesis is mediated by SYMRK phosphorylation, which may help when engineering this trait into non-legume plants.

The soil emergence-related transcription factor PIF3 controls root penetration by interacting with the receptor kinase FER.

The cotyledons of etiolated seedlings from terrestrial flowering plants must emerge from the soil surface, while roots must penetrate the soil to ensure plant survival. We show here that the soil emergence-related transcription factor PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) controls root penetration via transducing external signals perceived by the receptor kinase FERONIA (FER) in Arabidopsis thaliana. The loss of FER function in Arabidopsis and soybean (Glycine max) mutants resulted in a severe defect in root penetration into agar medium or hard soil. Single-cell RNA sequencing (scRNA-seq) profiling of Arabidopsis roots identified a distinct cell clustering pattern, especially for root cap cells, and identified PIF3 as a FER-regulated transcription factor. Biochemical, imaging, and genetic experiments confirmed that PIF3 is required for root penetration into soil. Moreover, FER interacted with and stabilized PIF3 to modulate the expression of mechanosensitive ion channel PIEZO and the sloughing of outer root cap cells.

Growth-inhibiting effects of the unconventional plant APYRASE 7 of Arabidopsis thaliana influences the LRX/RALF/FER growth regulatory module.

Plant cell growth involves coordination of numerous processes and signaling cascades among the different cellular compartments to concomitantly enlarge the protoplast and the surrounding cell wall. The cell wall integrity-sensing process involves the extracellular LRX (LRR-Extensin) proteins that bind RALF (Rapid ALkalinization Factor) peptide hormones and, in vegetative tissues, interact with the transmembrane receptor kinase FERONIA (FER). This LRX/RALF/FER signaling module influences cell wall composition and regulates cell growth. The numerous proteins involved in or influenced by this module are beginning to be characterized. In a genetic screen, mutations in Apyrase 7 (APY7) were identified to suppress growth defects observed in lrx1 and fer mutants. APY7 encodes a Golgi-localized NTP-diphosphohydrolase, but opposed to other apyrases of Arabidopsis, APY7 revealed to be a negative regulator of cell growth. APY7 modulates the growth-inhibiting effect of RALF1, influences the cell wall architecture and -composition, and alters the pH of the extracellular matrix, all of which affect cell growth. Together, this study reveals a function of APY7 in cell wall formation and cell growth that is connected to growth processes influenced by the LRX/RALF/FER signaling module.

The Klebsiella mannose phosphotransferase system promotes proliferation and the production of extracellular polymeric substances from mannose, facilitating adaptation to the host environment.

OBJECTIVES: Klebsiella spp., an opportunistic infectious organism, is commensal in the nasal and oral cavities of humans. Recently, it has been reported that oral Klebsiella spp. ectopically colonize the intestinal tract and induce the accumulation of intestinal Th1 cells. For oral bacteria to colonize the intestinal tract, they need to compete for nutrients with indigenous intestinal bacteria. Therefore, we focused on mannose, a mucus-derived sugar, and the mannose phosphotransferase system (PTS) (ManXYZ), a mechanism for mannose uptake, in terms of their effects on intestinal colonization and immune responses to Klebsiella spp. METHODS: We generated a Klebsiella manXYZ-deficient strain and investigated whether the utilization of intestinal mucus-derived sugars is associated with the growth. Furthermore, we examine the virulence of this organism in the mouse intestinal tract, especially the ability to colonize the host using competition assay. RESULTS: We found that Klebsiella ManXYZ is a PTS that specifically takes up mannose and glucosamine. Through ManXYZ, mannose was used for bacterial growth and the upregulated production of extracellular polymeric substances. In vivo competition assays showed that mannose metabolism promoted intestinal colonization. However, ManXYZ was not involved in Th1 and Th17 induction in the intestinal tract. CONCLUSION: The fundamental roles of ManXYZ were to ensure that mannose, which is present in the host, is made available for bacterial growth, to promote the production of extracellular polymeric substances, thus facilitating bacterial adaptation to the host environment.

Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development.

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2 F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2 F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2 F-based TCIs.

The tunicamycin derivative TunR2 exhibits potent antibiotic properties with low toxicity in an in vivo Mycobacterium marinum-zebrafish TB infection model.

Tunicamycins (TUN) are well-defined, Streptomyces-derived natural products that inhibit protein N-glycosylation in eukaryotes, and by a conserved mechanism also block bacterial cell wall biosynthesis. TUN inhibits the polyprenylphosphate-N-acetyl-hexosamine-1-phospho-transferases (PNPT), an essential family of enzymes found in both bacteria and eukaryotes. We have previously published the development of chemically modified TUN, called TunR1 and TunR2, that have considerably reduced activity on eukaryotes but that retain the potent antibacterial properties. A mechanism for this reduced toxicity has also been reported. TunR1 and TunR2 have been tested against mammalian cell lines in culture and against live insect cells but, until now, no in vivo evaluation has been undertaken for vertebrates. In the current work, TUN, TunR1, and TunR2 are investigated for their relative toxicity and antimycobacterial activity in zebrafish using a well-established Mycobacterium marinum (M. marinum) infection system, a model for studying human Mycobacterium tuberculosis infections. We also report the relative ability to activate the unfolded protein response (UPR), the known mechanism for the eukaryotic toxicity observed with TUN treatment. Importantly, TunR1 and TunR2 retained their antimicrobial properties, as evidenced by a reduction in M. marinum bacterial burden, compared to DMSO-treated zebrafish. In summary, findings from this study highlight the characteristics of recently developed TUN derivatives, mainly TunR2, and its potential for use as a novel anti-bacterial agent for veterinary and potential medical purposes.

Unveiling the fructose metabolism system in Staphylococcus aureus: insights into the regulatory role of FruR and the FruRKT operon in bacterial fitness.

BACKGROUND: The utilization of fructose as a carbon source and energy provider plays a crucial role in bacterial metabolism. Additionally, fructose metabolism directly impacts the pathogenicity and virulence of certain pathogenic microorganisms. RESULTS: In this study, we report the discovery of a fructose phosphotransferase system (PTS) in S. aureus. This system comprises three genes, namely fruR, fruK, and fruT, which are co-located in an operon that is indispensable for fructose utilization in S. aureus. Our findings confirm that these three genes are transcribed from a single promoter located upstream of the fruRKT operon. The fruR gene encodes a DeoR-type transcriptional regulator, designated as FruR, which represses the expression of the fruRKT operon by direct binding to its promoter region. Significantly, our experimental data demonstrate that the fruRKT operon can be induced by fructose, suggesting a potential regulatory mechanism involving intracellular fructose-1-phosphate as a direct inducer. Furthermore, we conducted RNA-seq analysis to investigate the specificity of FruR regulation in S. aureus, revealing that the fruRKT operon is predominantly regulated by FruR. CONCLUSIONS: In summary, this study has uncovered a fructose phosphotransferase system (PTS) in S. aureus, highlighting the essential role of the fruR, fruK, and fruT genes in fructose utilization. We confirmed their co-location within an operon and established FruR as a key regulator by binding to the operon's promoter. Importantly, we demonstrated that fructose can induce this operon, possibly through intracellular fructose-1-phosphate. Our identification of this PTS system represents the initial characterization of a fructose metabolism system in S. aureus.

Lactococcus lactis mutants resistant to lactococcin A and garvicin Q reveal missense mutations in the sugar transport domain of the mannose phosphotransferase system.

IMPORTANCE: Many bacteriocins target the sugar transporter mannose phosphotransferase system (man-PTS) to exert their antibacterial activity. The elucidation in recent years of the structure of man-PTS has facilitated our understanding of how bacteriocins might interact with the receptor and which domains of the transporter are involved in bacteriocin resistance. Here, we show that missense mutations in the sugar-binding domain of the man-PTS not only impede the uptake of sugars but also prevent the antibacterial activity of the bacteriocins lactococcin A and garvicin Q.

Novel synthetic biological study on intracellular distribution of human GlcNAc-1-phosphotransferase expressed in insect cells.

Many lysosomal enzymes contain N-glycans carrying mannose 6-phosphate (M6P) residues. Modifying lysosomal enzymes by M6P residues requires a two-step process in the Golgi apparatus. Then the lysosomal enzymes with M6P residues are transported from the trans-Golgi network to endosomes and lysosomes by M6P receptors. In insect cells, M6P residues are not added to N-glycans. Therefore, many insect lysosomal enzymes are transported to lysosomes by the M6P-independent pathway. The expression and subcellular distribution of M6P-modifying enzymes were examined by amplifying DNA fragments of M6P-modifying enzymes, generating the corresponding plasmid constructs, and transfection each construct into Sf9 cells, an insect cell line. The human GlcNac-1-phosphotransferase α/ß subunit, one of the M6P-modifying enzymes, was found to differ in maturation and localization between mammalian and insect cells. In mammalian cells, newly biosynthesized α/ß subunit localized in the cis-Golgi. In Sf9 cells, most of the α/ß subunit was localized in the endoplasmic reticulum, and few mature forms of α/ß subunit were observed. However, by the co-expression of the human site-1 protease, the mature forms were observed significantly and co-localization with each protein. Our study indicates new insights into regulating the intracellular distribution of the human GlcNac-1-phosphotransferase α/ß subunit in insect cells.

Ampelopsis japonica aqueous extract improves ovulatory dysfunction in PCOS by modulating lipid metabolism.

Polycystic ovary syndrome (PCOS) is a highly prevalent endocrine and metabolic disorder that is closely associated with the proliferation and apoptosis of ovarian granulosa cells (GCs). Ampelopsis japonica (AJ) is the dried tuberous root of Ampelopsis japonica (Thunb.) Makino (A. japonica), with anti-inflammatory, antioxidant, antibacterial, antiviral, wound-healing, and antitumor properties; however, it is unclear whether this herb has a therapeutic effect on PCOS. Therefore, this study aimed to investigate the pharmacological effect of AJ on PCOS and reveal its potential mechanism of action. A PCOS rat model was established using letrozole. After establishing the PCOS model, the rats received oral treatment of AJ and Diane-35 (Positive drug: ethinylestradiol + cyproterone tablets) for 2 weeks. Lipidomics was conducted using liquid-phase mass spectrometry and chromatography. AJ significantly regulated serum hormone levels and attenuated pathological variants in the ovaries of rats with PCOS. Furthermore, AJ significantly reduced the apoptotic rate of ovarian GCs. Lipidomic analysis revealed that AJ modulated glycerolipid and glycerophospholipid metabolic pathways mediated by lipoprotein lipase (Lpl), diacylglycerol choline phosphotransferase (Chpt1), and choline/ethanolamine phosphotransferase (Cept1). Therefore, we established that AJ may reduce ovarian GC apoptosis by modulating lipid metabolism, ultimately improving ovulatory dysfunction in PCOS. Therefore, AJ is a novel candidate for PCOS treatment.

Extracellular pectin-RALF phase separation mediates FERONIA global signaling function.

The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.

In vitro characterization of alternative l-threonate and d-erythronate catabolic pathways.

l-threonate is the metabolite of vitamin C, while d-erythronate is the metabolite of N-acetyl-d-glucosamine, the nutritional supplement for joint health. They are widely distributed in the environment and human biofluids. Nevertheless, the catabolisms of l-threonate and d-erythronate are sparsely reported. Here we explored the functional diversity of an acid sugar kinase family (Pfam families PF07005-PF17042), and discovered a novel 2-oxo-tetronate kinase. The conserved genome neighborhood of the 2-oxo-tetronate kinase encodes members of class-II fructose-bisphosphate aldolase family (F_bP_aldolase, PF01116) and a dehydrogenase family (PF03446-PF14833). Instructed by this analysis, we experimentally verified that these enzymes are capable of degrading l-threonate into dihydroxyacetone phosphate (DHAP) in Arthrobacter sp. ZBG10, Clostridium scindens ATCC 35704, and Pseudonocardia dioxanivorans ATCC 55486. Meanwhile, a convergent catabolic pathway for d-erythronate was characterized in P. dioxanivorans ATCC 55486. Moreover, the phylogenetic distribution analysis indicates that the biological range of the identified l-threonate and d-erythronate catabolic pathways appears to extend mostly to members of the Actinomycetota, Cyanobacteriota, Bacillota, Pseudomonadota, and Bacteroidota phyla.