Pectin methylesterase activity is required for RALF1 peptide signalling output.

The extracellular matrix plays an integrative role in cellular responses in plants, but its contribution to the signalling of extracellular ligands largely remains to be explored. Rapid alkalinisation factors (RALFs) are extracellular peptide hormones that play pivotal roles in various physiological processes. Here, we address a crucial connection between the de-methylesterification machinery of the cell wall component pectin and RALF1 activity. Pectin is a polysaccharide, contributing to the structural integrity of the cell wall. Our data illustrate that the pharmacological and genetic interference with pectin methyl esterases (PMEs) abolishes RALF1-induced root growth repression. Our data suggest that positively charged RALF1 peptides bind negatively charged, de-methylesterified pectin with high avidity. We illustrate that the RALF1 association with de-methylesterified pectin is required for its FERONIA-dependent perception, contributing to the control of the extracellular matrix and the regulation of plasma membrane dynamics. Notably, this mode of action is independent of the FER-dependent extracellular matrix sensing mechanism provided by FER interaction with the leucine-rich repeat extensin (LRX) proteins. We propose that the methylation status of pectin acts as a contextualizing signalling scaffold for RALF peptides, linking extracellular matrix dynamics to peptide hormone-mediated responses.

Machine learning-enabled computer vision for plant phenotyping: a primer on AI/ML and case study on stomatal patterning.

Artificial intelligence and machine learning (AI/ML) can be used to automatically analyze large image datasets. One valuable application of this approach is estimation of plant trait data contained within images. Here we review 39 papers that describe the development and/or application of such models for estimation of stomatal traits from epidermal micrographs. In doing so, we hope to provide plant biologists with a foundational understanding of AI/ML and summarize the current capabilities and limitations of published tools. While most models show human-level performance for stomatal density (SD) quantification at superhuman speed, they are often likely to be limited in how broadly they can be applied across phenotypic diversity associated with genetic, environmental or developmental variation. Other models can make predictions across greater phenotypic diversity and/or additional stomatal/epidermal traits, but require significantly greater time investment to generate ground-truth data. We discuss the challenges and opportunities presented by AI/ML-enabled computer vision analysis, and make recommendations for future work to advance accelerated stomatal phenotyping.

Design and Characterization of a Transcriptional Repression Toolkit for Plants.

Regulation of gene expression is essential for all life. Tools to manipulate the gene expression level have therefore proven to be very valuable in efforts to engineer biological systems. However, there are few well-characterized genetic parts that reduce gene expression in plants, commonly known as transcriptional repressors. We characterized the repression activity of a library consisting of repression motifs from approximately 25% of the members of the largest known family of repressors. Combining sequence information with our trans-regulatory function data, we next generated a library of synthetic transcriptional repression motifs with function predicted in advance. After characterizing our synthetic library, we demonstrated not only that many of our synthetic constructs were functional as repressors but also that our advance predictions of repression strength were better than random guesses. Finally, we assessed the functionality of known transcriptional repression motifs from a wide range of eukaryotes. Our study represents the largest plant repressor motif library experimentally characterized to date, providing unique opportunities for tuning transcription in plants.

Genome of the most noxious weed water hyacinth (Eichhornia crassipes) provides insights into plant invasiveness and its translational potential.

The invasive character of Eichhornia crassipes (water hyacinth) is a major threat to global biodiversity and ecosystems. To investigate the genomic basis of invasiveness, we performed the genome and transcriptome sequencing of E. crassipes and reported the genome of 1.11 Gbp size with 63,299 coding genes and N50 of 1.98 Mb. We confirmed a recent whole genome duplication event in E. crassipes that resulted in high intraspecific collinearity and significant expansion in gene families. Further, the orthologs gene clustering analysis and comparative evolutionary analysis with 14 other aquatic invasive and non-invasive angiosperm species revealed adaptive evolution in genes associated with plant-pathogen interaction, hormone signaling, abiotic stress tolerance, heavy metals sequestration, photosynthesis, and cell wall biosynthesis with highly expanded gene families, which contributes toward invasive characteristics of the water hyacinth. However, these characteristics also make water hyacinth an excellent candidate for biofuel production, phytoremediation, and other translational applications.

Single-cell landscape of long and short glandular trichomes in Nicotiana tabacum leaves.

Glandular trichomes (GTs) play a crucial role in plant defenses and the synthesis of secondary metabolites. Understanding the developmental trajectory of GTs is essential for unraveling their functional significance and potential applications. Here we established a comprehensive single-cell atlas of Nicotiana tabacum leaves, a model plant for GT studies. The atlas included a total of 40,433 cells and successfully captured both long GTs (LGTs) and short GTs (SGTs) from Nicotiana leaves. The developmental trajectories of these trichomes were delineated, revealing potential disparities in epidermal development. Comparative analysis of Arabidopsis and Nicotiana trichome development indicated limited similarity between Arabidopsis epidermal non-glandular trichomes and Nicotiana LGTs and SGTs, implying the essentiality of studying the genes directly involved in the development of Nicotiana GTs for a proper and comprehensive understanding of GT biology. Overall, our results provide profound insights into the developmental intricacies of the specialized GTs.

Widespread application of apomixis in agriculture requires further study of natural apomicts.

Apomixis, or asexual reproduction through seeds, is frequent in nature but does not exist in any major crop species, yet the phenomenon has captivated researchers for decades given its potential for clonal seed production and plant breeding. A discussion on whether this field will benefit from the continued study of natural apomicts is warranted given the recent outstanding progress in engineering apomixis. Here, we summarize what is known about its genetic control and the status of applying synthetic apomixis in agriculture. We argue there is still much to be learned from natural apomicts, and learning from them is necessary to improve on current progress and guarantee the effective application of apomixis beyond the few genera it has shown promise in so far. Specifically, we stress the value of studying the repeated evolution of natural apomicts in a phylogenetic and comparative -omics context. Finally, we identify outstanding questions in the field and discuss how technological advancements can be used to help close these knowledge gaps. In particular, genomic resources are lacking for apomicts, and this must be remedied for widespread use of apomixis in agriculture.

Structure and evolution of alanine/serine decarboxylases and the engineering of theanine production.

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr341 of AtSerDC or the corresponding Tyr336 of CsAlaDC is essential for their enzymatic activity. Tyr111 of AtSerDC and the corresponding Phe106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDCL110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine.

Evolution of phosphate scouting in the terrestrial biosphere.

Chemistry assigns phosphorus and its most oxidized form, inorganic phosphate, unique roles for propelling bioenergetics and metabolism in all domains of life, possibly since its very origin on prebiotic Earth. For plants, access to the vital mineral nutrient profoundly affects growth, development and vigour, thus constraining net primary productivity in natural ecosystems and crop production in modern agriculture. Unlike other major biogenic elements, the low abundance and uneven distribution of phosphate in Earth's crust result from the peculiarities of phosphorus cosmochemistry and geochemistry. Here, we trace the chemical evolution of the element, the geochemical phosphorus cycle and its acceleration during Earth's history until the present (Anthropocene) as well as during the evolution and rise of terrestrial plants. We highlight the chemical and biological processes of phosphate mobilization and acquisition, first evolved in bacteria, refined in fungi and algae and expanded into powerful phosphate-prospecting strategies during land plant colonization. Furthermore, we review the evolution of the genetic and molecular networks from bacteria to terrestrial plants, which monitor intracellular and extracellular phosphate availabilities and coordinate the appropriate responses and adjustments to fluctuating phosphate supply. Lastly, we discuss the modern global phosphorus cycle deranged by human activity and the challenges imposed ahead. This article is part of the theme issue 'Evolution and diversity of plant metabolism'.

A reduced vernalization requirement is a key component of the early-bolting trait in globe artichoke (Cynara cardunculus var. scolymus).

Early bolting is a major breeding objective for globe artichoke (Cynara cardunculus var. scolymus L.). It has been suggested that globe artichoke bolting time is linked to a vernalization requirement, although environmental conditions under which vernalized plants and controls have been grown may not always allow for proper comparison. Here, we defined morphological markers to monitor the vegetative-to-reproductive phase transition at the shoot apex and linked these to expression changes of homologs of key Arabidopsis flowering regulators SOC1, FUL, and AP1. Importantly, we developed an experimental setup where control and vernalized plants grow under comparable conditions. These tools together allowed for comparison of the vegetative-to-reproductive phase transition between early- and late-bolting genotypes and how they respond to vernalization. Our results show that vernalization requirement is significantly lower in early-bolting genotypes, supporting the hypothesis that the early-bolting trait is at least partly underlain by alterations in the network controlling vernalization response.

FYVE1/FREE1 is involved in glutamine-responsive TORC1 activation in plants.

Target of rapamycin complex 1 (TORC1) integrates nutrient availability, growth factors, and stress signals to regulate cellular metabolism according to its environment. Similar to mammals, amino acids have been shown to activate TORC1 in plants. However, as the Rag complex that controls amino acid-responsive TORC1 activation mechanisms in many eukaryotes is not conserved in plants, the amino acid-sensing mechanisms upstream of TORC1 in plants remain unknown. In this study, we report that Arabidopsis FYVE1/FREE1 is involved in glutamine-induced TORC1 activation, independent of its previously reported function in ESCRT-dependent processes. FYVE1/FREE1 has a domain structure similar to that of the yeast glutamine sensor Pib2 that directly activates TORC1. Similar to Pib2, FYVE1/FREE1 interacts with TORC1 in response to glutamine. Furthermore, overexpression of a FYVE1/FREE1 variant lacking the presumptive TORC1 activation motif hindered the glutamine-responsive activation of TORC1. Overall, these observations suggest that FYVE1/FREE1 acts as an intracellular amino acid sensor that triggers TORC1 activation in plants.

Deciphering sphingolipid biosynthesis dynamics in Arabidopsis thaliana cell cultures: Quantitative analysis amid data variability.

Sphingolipids are pivotal for plant development and stress responses. Growing interest has been directed toward fully comprehending the regulatory mechanisms of the sphingolipid pathway. We explore its de novo biosynthesis and homeostasis in Arabidopsis thaliana cell cultures, shedding light on fundamental metabolic mechanisms. Employing 15N isotope labeling and quantitative dynamic modeling approach, we obtained data with notable variations and developed a regularized and constraint-based dynamic metabolic flux analysis (r-DMFA) framework to predict metabolic shifts due to enzymatic changes. Our analysis revealed key enzymes such as sphingoid-base hydroxylase (SBH) and long-chain-base kinase (LCBK) to be critical for maintaining sphingolipid homeostasis. Disruptions in these enzymes were found to affect cellular viability and increase the potential for programmed cell death (PCD). Despite challenges posed by data variability, this work enhances our understanding of sphingolipid metabolism and demonstrates the utility of dynamic modeling in analyzing complex metabolic pathways.

OsNF-YB7 inactivates OsGLK1 to inhibit chlorophyll biosynthesis in rice embryo.

As a master regulator of seed development, Leafy Cotyledon 1 (LEC1) promotes chlorophyll (Chl) biosynthesis in Arabidopsis, but the mechanism underlying this remains poorly understood. Here, we found that loss of function of OsNF-YB7, a LEC1 homolog of rice, leads to chlorophyllous embryo, indicating that OsNF-YB7 plays an opposite role in Chl biosynthesis in rice compared with that in Arabidopsis. OsNF-YB7 regulates the expression of a group of genes responsible for Chl biosynthesis and photosynthesis by directly binding to their promoters. In addition, OsNF-YB7 interacts with Golden 2-Like 1 (OsGLK1) to inhibit the transactivation activity of OsGLK1, a key regulator of Chl biosynthesis. Moreover, OsNF-YB7 can directly repress OsGLK1 expression by recognizing its promoter in vivo, indicating the involvement of OsNF-YB7 in multiple regulatory layers of Chl biosynthesis in rice embryo. We propose that OsNF-YB7 functions as a transcriptional repressor to regulate Chl biosynthesis in rice embryo.

Impact of zinc on arbuscular mycorrhizal-mediated nutrient acquisition in urban horticulture.

A major barrier to sustainably improving food security for a growing global population is the availability of suitable space for growing crops. Urban areas offer a potential solution to increase availability of land, however, horticultural soils often accumulate zinc. These increased levels may affect the interactions between crops and soil microbes with potential implications for crop health and nutrition. Using radio-isotope tracing, we investigated the effect of urban environmentally relevant concentrations of zinc in soils on the nutrient exchange between arbuscular mycorrhizal fungi and pea plants. At higher concentrations of zinc, transfer of phosphorus from fungi to plants and the movement of carbon from plants to fungi was dramatically decreased. Our results suggest that while urban horticulture holds promise for sustainably enhancing local food production and addressing global food security, the unchecked presence of contaminants in these soils may pose a critical hurdle to realizing the potential of urban soils.

Coping with salt stress.

Salt stress delays seed germination in plants by increasing the hydrolysis of arginine-derived urea.

Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding protein gene for canker formation.

Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1. This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620, which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1.

The genetic architecture of the pepper metabolome and the biosynthesis of its signature capsianoside metabolites.

Capsicum (pepper) is among the most economically important species worldwide, and its fruits accumulate specialized metabolites with essential roles in plant environmental interaction and human health benefits as well as in conferring their unique taste. However, the genetics underlying differences in metabolite presence/absence and/or accumulation remain largely unknown. In this study, we carried out a genome-wide association study as well as generating and characterizing a novel backcross inbred line mapping population to determine the genetic architecture of the pepper metabolome. This genetic analysis provided over 1,000 metabolic quantitative trait loci (mQTL) for over 250 annotated metabolites. We identified 92 candidate genes involved in various mQTLs. Among the identified loci, we described and validated a gene cluster of eleven UDP-glycosyltransferases (UGTs) involved in monomeric capsianoside biosynthesis. We additionally constructed the gene-by-gene-based biosynthetic pathway of pepper capsianoside biosynthesis, including both core and decorative reactions. Given that one of these decorative pathways, namely the glycosylation of acyclic diterpenoid glycosides, contributes to plant resistance, these data provide new insights and breeding resources for pepper. They additionally provide a blueprint for the better understanding of the biosynthesis of species-specific natural compounds in general.

Pleiotropic regulatory locus 1 maintains actin cytoskeleton integrity and cellular homeostasis to enable Arabidopsis root growth.

Cell functions are based on the integrity of actin filaments. The actin cytoskeleton is typically the target but also the source of signals. Arabidopsis PRL1 (Pleiotropic Regulatory Locus 1), regulates multiple cellular processes and physiological responses. However, the precise mechanisms underlying PRL1`s multiple functions are unclear. Here, we show that PRL1 maintains actin integrity and concomitant cellular homeostasis. The cortical actin cytoskeleton was de-polymerized in the prl1 mutant, causing the developmental root defect. Actin depolymerization, rather than reactive oxygen species (ROS) imbalance, constituted the fundamental cause of retarded root growth in prl1. ANAC085 upregulation by, and cooperation with, actin depolymerization triggered stele cell death in prl1 roots. Differential gene expression and alternative splicing defects resulting from actin depolymerization occurred independently in prl1. Our work establishes the cause-effect relationships between actin depolymerization and downstream stress-related signals, revealing a novel function of PRL1 and enhancing the understanding of PRL`s functional mechanisms.

The cell cycle controls spindle architecture in Arabidopsis by activating the augmin pathway.

To ensure an even segregation of chromosomes during somatic cell division, eukaryotes rely on mitotic spindles. Here, we measured prime characteristics of the Arabidopsis mitotic spindle and built a three-dimensional dynamic model using Cytosim. We identified the cell-cycle regulator CYCLIN-DEPENDENT KINASE B1 (CDKB1) together with its cyclin partner CYCB3;1 as key regulators of spindle morphology in Arabidopsis. We found that the augmin component ENDOSPERM DEFECTIVE1 (EDE1) is a substrate of the CDKB1;1-CYCB3;1 complex. A non-phosphorylatable mutant rescue of ede1 resembled the spindle phenotypes of cycb3;1 and cdkb1 mutants and the protein associated less efficiently with spindle microtubules. Accordingly, reducing the level of augmin in simulations recapitulated the phenotypes observed in the mutants. Our findings emphasize the importance of cell-cycle-dependent phospho-control of the mitotic spindle in plant cells and support the validity of our model as a framework for the exploration of mechanisms controlling the organization of the eukaryotic spindle.

Indole-3-propionic acid regulates lateral root development by targeting auxin signaling in Arabidopsis.

Indole-3-propionic acid (IPA) is known to be a microbe-derived compound with a similar structure to the phytohormone auxin (indole-3-acetic acid, IAA). Previous studies reported that IPA exhibited auxin-like bioactivities in plants. However, the underlying molecular mechanism is not totally understood. Here, we revealed that IPA modulated lateral root (LR) development via auxin signaling in the model plant Arabidopsis thaliana. Genetic analysis indicated that deficiency of the TIR1/AFB-Aux/IAA-ARF auxin signaling pathway abolished the effects of IPA on regulating LR development. Further biochemical, transcriptomic profiling and cell biological analyses revealed that IPA directly bound to the TIR1/AFB-Aux/IAA coreceptor complex and thus activated downstream gene expression. Therefore, our work revealed that IPA is a potential signaling molecule that modulates plant growth and development by targeting the TIR1/AFB-Aux/IAA-mediated auxin signaling pathway, providing potential insights into root growth regulation in plants.