Species occupying broad geographic regions have evolved multiple mechanisms to regulate phenological characteristics, enabling adaptations to diverse native habitats. By developing computer vision AI to process citizen science observations across native habitats over North America, we uncovered a consistent latitudinal trend of earlier flowering at higher latitudes in warm-season perennial grasses. To explore the underlying mechanisms of adaptation, we conducted common garden experiments with one species (switchgrass) and discovered the opposite latitudinal flowering-time trend. Integration of differential plasticity of GI-Hd1-FTL1 haplotypes of flowering time regulatory genes, haplotype range, and local environmental profiles found that observations from native habitats capture only part of the genotype-environment-phenotype spectrum established in common garden experiments, therefore reconciling the discrepancy. Two mechanisms emerged as key forces shaping current haplotype ranges and influencing future shifts. Our study highlights the power of combining citizen science observations with designed experiments to uncover mechanisms of adaptation across spatiotemporal scales.

Abstract Plant root cellular architecture and cell wall composition influence plant productivity, stress resilience, biotic interactions, and potentially soil carbon accumulation. This study establishes comprehensive compositional parameters for roots of a lowland switchgrass genotype, DVR3. Root traits were analyzed in 12.5 cm depth segments, from Zone 1 near the surface to Zone 4 down to 50 cm. Mean abundance (µg/mg) for major cell wall components included cellulose 470 ± 20, xylose 250 ± 20, lignin 170 ± 15, and total suberin 35 ± 5. Composition and cellular anatomy varied with depth, in a partially coordinated manner. Cross sections showed extensive aerenchyma in mature root regions despite greater root mass density, corresponding to abundant lignin and cellulose. Deep roots were enriched for pectin-associated traits, including arabinogalactan II, homogalacturonan, and arabinose-associated linkages. Suberin content did not vary significantly, though Casparian strip formation, endoderm and exoderm thickening, and suberin surface staining progressed with development. Similar trends in root lignin and specific root length were observed for another lowland switchgrass genotype, AP13. These results suggest that it may be possible to genetically enhance native switchgrass root chemistry to promote soil penetration and below-ground carbon accumulation by reducing variability with development, potentially via cell-type specific adjustments. Highlight Older, shallower switchgrass crown roots are enriched in lignin and cellulose, and deeper, younger roots are pectin-rich with juvenile cellular anatomy. A more uniform compositional distribution might enhance below-ground traits.

This beginner's guide is intended for plant biologists new to network analysis. Here, we introduce key concepts and resources for researchers interested in incorporating network analysis into research, either as a stand-alone component for generating hypotheses or as a framework for examining and visualizing experimental results. Network analysis provides a powerful tool to predict gene functions. Advances in and reduced costs for systems biology techniques, such as genomics, transcriptomics, and proteomics, have generated abundant omics data for plants; however, the functional annotation of plant genes lags. Therefore, predictions from network analysis can be a starting point to annotate genes and ultimately elucidate genotype-phenotype relationships. In this paper, we introduce networks and compare network-building resources available for plant biologists, including databases and software for network analysis. We then compare four databases available for plant biologists in more detail: AraNet, GeneMANIA, ATTED-II, and STRING. AraNet and GeneMANIA are functional association networks, ATTED-II is a gene coexpression database, and STRING is a protein-protein interaction database. AraNet and ATTED-II are plant-specific databases that can analyze multiple plant species, whereas GeneMANIA builds networks for Arabidopsis thaliana and nonplant species and STRING for multiple species. Finally, we compare the performance of the four databases in predicting known and probable gene functions of the A. thaliana Nuclear Factor-Y (NF-Y) genes. We conclude that plant biologists have an invaluable resource in these databases and discuss how users can decide which type of database to use depending on their research question.

Extracellular ATP (eATP) signaling in Arabidopsis thaliana is mediated by the purinoceptor P2K1. Previous studies have clarified that the downstream transcriptional responses to eATP involve jasmonate (JA)-based signaling components such as the JA receptor (COI1) and JA-responsive bHLH transcription factors (MYCs). However, the specific contributions of JA itself on eATP signaling are unexplored. Here, we report that JA primes plant responses to eATP through P2K1. Our findings show that JA treatment significantly upregulates P2K1 transcription, corroborating our observation that JA facilitates eATP-induced cytosolic calcium elevation and transcriptional reprogramming in a JA signaling-dependent manner. Additionally, we find that salicylic acid pre-treatment represses eATP-induced plant response. These results suggest that JA accumulation during biotic or abiotic stresses potentiates eATP signaling, enabling plants to better cope with subsequent stress events.

Cell wall composition influences biomass use as a forage and as a feedstock for biofuel and chemical conversion. To examine the influence of environment on composition of switchgrass (Panicum virgatum L.), we utilized a multi-environment experiment consisting of clones of switchgrass genotypes grown at up to ten locations in the continental US. We tested the influence of different milling treatments on biomass composition trait predictions via near-infrared reflectance spectroscopy (NIRS). We found that most compositional trait predictions (29/34) were significantly different (P < 0.05) when a single lot of biomass was subjected to disparate milling treatments, i.e., knife milling vs. knife milling with an additional cyclone milling. Further, depending on the plant material tested, three to eight compositional trait predictions vary (P < 0.05) when identical biomass was knife milled at different sites followed by cyclone milling at a single site, including for traits such as Klason lignin, nitrogen, and carbon. In some cases, variation due to milling site exceeded environmentally induced compositional variation of a single switchgrass genotype grown at different sites. From these observations, we recommend a protocol with two sequential millings that decouples growth environment from a particular mill. Utilizing this approach, we found that 46/46 biomass composition traits from the warm season herbaceous forage and switchgrass bioethanol NIRS equations vary significantly (P < 0.001) in clones of a switchgrass genotype (WBC) grown at ten sites, with the growth site representing the largest average source of variation (41

In eukaryotic organisms, protein kinases regulate diverse protein activities and signaling pathways through phosphorylation of specific protein substrates. Isolating and characterizing kinase substrates is vital for defining downstream signaling pathways. The Kinase Client (KiC) assay is an in vitro synthetic peptide LC-MS/MS phosphorylation assay that has enabled identification of protein substrates (i.e., clients) for various protein kinases. For example, previous use of a 2,100-member (2k) peptide library identified substrates for the extracellular ATP receptor-like kinase, P2K1. Many P2K1 clients were confirmed by additional in vitro and in planta studies, including Integrin-Linked Kinase 4 (ILK4), for which we provide the evidence herein. In addition, we developed a new KiC peptide library containing 8,000 (8k) peptides based on phosphorylation sites primarily from Arabidopsis thaliana datasets. The 8k peptides are enriched for sites with conservation in other angiosperm plants, with the paired goals of representing functionally conserved sites and usefulness for screening kinases from diverse plants. Screening the 8k library with the active P2K1 kinase domain identified 177 phosphopeptides, including calcineurin B-like protein (CBL9) and G protein alpha subunit 1 (GPA1), which functions in cellular calcium signaling. We confirmed that P2K1 directly phosphorylates CBL9 and GPA1 through in vitro kinase assays. This expanded 8k KiC assay will be a useful tool for identifying novel substrates across diverse plant protein kinases, ultimately facilitating the exploration of previously undiscovered signaling pathways.

Lignin, flavonoids, melatonin, and stilbenes are plant specialized metabolites with diverse physiological and biological functions, supporting plant growth and conferring stress resistance. Their biosynthesis requires O-methylations catalyzed by 5-hydroxyconiferaldehyde O-methyltransferase (CAldOMT; also called caffeic acid O-methyltransferase, COMT). CAldOMT was first known for its roles in syringyl (S) lignin biosynthesis in angiosperm cell walls and later found to be multifunctional. This enzyme also catalyzes O-methylations in flavonoid, melatonin, and stilbene biosynthetic pathways. Phylogenetic analysis indicated the convergent evolution of enzymes with OMT activities towards the monolignol biosynthetic pathway intermediates in some gymnosperm species that lack S-lignin and Selaginella moellendorffii, a lycophyte which produces S-lignin. Furthermore, neofunctionalization of CAldOMTs occurred repeatedly during evolution, generating unique O-methyltransferases (OMTs) with novel catalytic activities and/or accepting novel substrates, including lignans, 1,2,3-trihydroxybenzene, and phenylpropenes. This review summarizes multiple aspects of CAldOMTs and their related proteins in plant metabolism and discusses their evolution, molecular mechanism, and roles in biorefineries, agriculture, and synthetic biology.

The phenolic polymer lignin is one of the primary chemical constituents of the plant secondary cell wall. Due to the inherent plasticity of lignin biosynthesis, several phenolic monomers have been shown to be incorporated into the polymer, as long as the monomer can undergo radicalization so it can participate in coupling reactions. In this study, we significantly enhance the level of incorporation of monolignol ferulate conjugates into the lignin polymer to improve the digestibility of lignocellulosic biomass. Overexpression of a rice Feruloyl-CoA Monolignol Transferase (FMT), OsFMT1, in hybrid poplar (Populus alba x grandidentata) produced transgenic trees clearly displaying increased cell wall-bound ester-linked ferulate, p-hydroxybenzoate, and p-coumarate, all of which are in the lignin cell wall fraction, as shown by NMR and DFRC. We also demonstrate the use of a novel UV–Vis spectroscopic technique to rapidly screen plants for the presence of both ferulate and p-hydroxybenzoate esters. Lastly we show, via saccharification assays, that the OsFMT1 transgenic p oplars have significantly improved processing efficiency compared to wild-type and Angelica sinensis-FMT-expressing poplars. The findings demonstrate that OsFMT1 has a broad substrate specificity and a higher catalytic efficiency compared to the previously published FMT from Angelica sinensis (AsFMT). Importantly, enhanced wood processability makes OsFMT1 a promising gene to optimize the composition of lignocellulosic biomass.

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants’ cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries.

In grass cell walls, ferulic acid (FA) serves as an important cross-linker between cell wall polymers, such as arabinoxylan (AX) and lignin, affecting the physicochemical properties of the cell walls as well as the utilization properties of grass lignocellulose for biorefinering. Here, we demonstrate that hydroxycinnamaldehyde dehydrogenase (HCALDH) plays a crucial role in the biosynthesis of the FA used for cell wall feruloylation in rice (Oryza sativa). Bioinformatic and gene expression analyses of aldehyde dehydrogenases (ALDHs) identified two rice ALDH subfamily 2C members, OsHCALDH2 (OsALDH2C2) and OsHCALDH3 (OsALDH2C3), potentially involved in cell wall feruloylation in major vegetative tissues of rice. CRISPR-Cas9 genome editing of OsHCALDH2 and OsHCALDH3 revealed that the contents of AX-bound ferulate were reduced by up to ~45% in the cell walls of the HCALDH-edited mutants, demonstrating their roles in cell wall feruloylation. The abundance of hemicellulosic sugars including arabinosyl units on AX was notably reduced in the cell walls of the HCALDH-edited mutants, whereas cellulose and lignin contents remained unaffected. In addition to reducing cell wall-bound ferulate, the loss of OsHCALDH2 and/or OsHCALDH3 also partially reduced cell wall-bound p-coumarate and sinapate in the vegetative tissues of rice, whereas it did not cause detectable changes in the amount of γ-oryzanol (feruloyl sterols) in rice seeds. Furthermore, the HCALDH-edited mutants exhibited improved cell wall saccharification efficiency, both with and without alkaline pretreatment, plausibly due to the reduction in cell wall cross-linking FA. Overall, HCALDH appears to present a potent bioengineering target for enhancing utilization properties of grass lignocellulose.

Extracellular ATP (eATP) signaling in Arabidopsis thaliana is mediated by the purinoceptor P2K1. Previous studies have clarified that the downstream transcriptional responses to eATP involve jasmonate (JA)-based signaling components such as the JA receptor (COI1) and JA-responsive bHLH transcription factors (MYCs). However, the specific contributions of JA signaling itself on eATP signaling are unexplored. Here, we report that JA primes plant responses to eATP through P2K1. Our findings show that JA treatment significantly upregulates P2K1 transcription, corroborating our observation that JA facilitates eATP-induced cytosolic calcium elevation and transcriptional reprogramming in a JA signaling-dependent manner. Additionally, we find that salicylic acid represses eATP-induced plant response. These results suggest that JA accumulation during biotic or abiotic stresses may potentiate eATP signaling, enabling plants to better cope with subsequent stress events. ### Competing Interest Statement The authors have declared no competing interest.

Plants are remarkable in their ability to adapt to changing environments, with receptor-like kinases (RLKs) playing a pivotal role in perceiving and transmitting environmental cues into cellular responses. Despite extensive research on RLKs from the plant kingdom, the function and activity of many kinases, i.e., their substrates or “clients”, remain uncharted. To validate a novel client prediction workflow and learn more about an important RLK, this study focuses on P2K1 (DORN1), which acts as a receptor for extracellular ATP (eATP), playing a crucial role in plant stress resistance and immunity. We designed a Kinase-Client (KiC) assay library of 225 synthetic peptides, incorporating previously identified P2K phosphorylated peptides and novel predictions from a deep-learning phosphorylation site prediction model (MUsite) and a trained hidden Markov model (HMM) based tool, HMMER. Screening the library against purified P2K1 cytosolic domain (CD), we identified 46 putative substrates, including 34 novel clients, 27 of which may be novel peptides, not previously identified experimentally. Gene Ontology (GO) analysis among phosphopeptide candidates revealed proteins associated with important biological processes in metabolism, structure development, and response to stress, as well as molecular functions of kinase activity, catalytic activity, and transferase activity. We offer selection criteria for efficient further in vivo experiments to confirm these discoveries. This approach not only expands our knowledge of P2K1’s substrates and functions but also highlights effective prediction algorithms for identifying additional potential substrates. Overall, the results support use of the KiC assay as a valuable tool in unraveling the complexities of plant phosphorylation and provide a foundation for predicting the phosphorylation landscape of plant species based on peptide library results.

Lignin is a phenolic heteropolymer found in most terrestrial plants that contributes an essential role in plant growth, abiotic stress tolerance, and biotic stress resistance. Recent research in grass lignin biosynthesis has found differences compared to dicots such as Arabidopsis thaliana. For example, the prolific incorporation of hydroxycinnamic acids into grass secondary cell walls improve the structural integrity of vascular and structural elements via covalent crosslinking. Conversely, fundamental monolignol chemistry conserves the mechanisms of monolignol translocation and polymerization across the plant phylum. Emerging evidence suggests grass lignin compositions contribute to abiotic stress tolerance, and periods of biotic stress often alter cereal lignin compositions to hinder pathogenesis. This same recalcitrance also inhibits industrial valorization of plant biomass, making lignin alterations and reductions a prolific field of research. This review presents an update of grass lignin biosynthesis, translocation, and polymerization, highlights how lignified grass cell walls contribute to plant development and stress responses, and briefly addresses genetic engineering strategies that may benefit industrial applications.

Switchgrass can generate large amounts of renewable biomass and hence is one of the most promising bioenergy crops. Improving the quality of switchgrass lignocellulosic biomass will enable its utilization for biofuels. Arabidopsis SHINE family of transcription factor SHN2 was previously identified as a master regulator of cell wall deposition in transgenic rice. However, it is unclear if the Arabidopsis SHN genes also have a similar biological function in switchgrass. Here, we generated transgenic switchgrass overexpressing the Arabidopsis SHN3 transcription factor. Compared with the wild-type, AtSHN3-overexpressing switchgrass plants were stunted in their growth. There were no significant differences in terms of lignin and cellulose content between the SHN transgenics and wild-type switchgrass plants. However, two AtSHN3 transgenic lines SHN7-2 and SHN5-2, displayed significant changes in several matrix polysaccharide monomers. Overexpression of AtSHN3 in switchgrass did not alter the stem mechanical strength when subjected to tensile-torsion analysis. Interestingly, the AtSHN3-overexpressing transgenic lines were more susceptible to switchgrass rust ( Puccinia emaculata) than wild-type plants. Therefore, AtSHN3 may have a negative role in regulating disease resistance in switchgrass.

Background Microscopic analysis of plant anatomy is a common procedure in biology to study structure and function that requires high-quality sections for accurate measurements. Hand sectioning of specimens is typically limited to moderately soft tissue while harder samples prohibit sectioning by hand and/or result in inconsistent thicknesses. Results Here we present both a clearly described hand-sectioning method and a novel microtome design that together provide the means to section a variety of plant sample types. The described hand-sectioning method for herbaceous stems works well for softer subjects but is less suitable for samples with secondary growth (e.g., wood production). Instead, the “Rapid-Tome” is a novel tool for sectioning both soft and tougher high-aspect-ratio samples, such as stems and roots, with excellent sample control. The Rapid-Tome can be 3D-printed in approximately 18 h on a mid-quality printer common at university maker spaces. After printing and trimming, Rapid-Tome assembly takes a few minutes with five metal parts common at hardware stores. Users sectioned a variety of plant samples including the hollow internodes of switchgrass ( Panicum virgatum ), fibrous switchgrass roots containing aerenchyma, and woody branches of eastern red cedar ( Juniperus virginiana ) and American sycamore ( Platanus occidentalis ). A comparative analyses with Rapid-Tome-produced sections readily revealed a significant difference in seasonal growth of sycamore xylem vessel area in spring (49%) vs. summer (23%). Additionally, high school students with no prior experience produced sections with the Rapid-Tome adequate for comparative analyses of various plant samples in less than an hour. Conclusions The described hand-sectioning method is suitable for softer tissues, including hollow-stemmed grasses and similar samples. In addition, the Rapid-Tome provides capacity to safely produce high-quality sections of tougher plant materials at a fraction of the cost of traditional microtomes combined with excellent sample control. The Rapid-Tome features rapid sectioning, sample advancement, blade changes, and sample changes; it is highly portable and can be used easily with minimal training making production of thin sections accessible for classroom and outreach use, in addition to research.

[This corrects the article DOI: 10.3389/fpls.2022.1088879.].

The cell cycle controls division and proliferation of all eukaryotic cells and is tightly regulated at multiple checkpoints by complexes of core cell cycle proteins. Due to the difficulty in accessing female gametes and zygotes of flowering plants, little is known about the molecular mechanisms underlying embryogenesis initiation despite the crucial importance of this process for seed crops. In this study, we reveal three levels of factors involved in rice zygotic cell cycle control and characterize their functions and regulation. Protein–protein interaction studies, including within zygote cells, and in vitro biochemical analyses delineate a model of the zygotic cell cycle core complex for rice. In this model, CDKB1, a major regulator of plant mitosis, is a cyclin (CYCD5)-dependent kinase; its activity is coordinately inhibited by two cell cycle inhibitors, KRP4 and KRP5; and both KRPs are regulated via F-box protein 3 (FB3)-mediated proteolysis. Supporting their critical roles in controlling the rice zygotic cell cycle, mutations in KRP4, KRP5 and FB3 result in the compromised function of sperm cells and abnormal organization of female germ units, embryo and endosperm, thus significantly reducing seed-set rate. This work helps reveal regulatory mechanisms controlling the zygotic cell cycle toward seed formation in angiosperms.

Cell wall properties can play a major role in determining photosynthetic carbon-uptake and water-use through impacts on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue to stomata). Consequently, modification of cell wall properties is proposed as a major path for improving photosynthesis and crop water-use efficiency. We tested this using two independent transgenic rice lines that overexpress the rice OsAT10 gene (a “BAHD” CoA acyltransferase) which altered cell wall hydroxycinnamic acid content (greater para -coumaric acid and lower ferulic acid). Plants were grown under high and low water-levels and traits related to leaf anatomy, cell wall composition, gas exchange and hydraulics, plant biomass, and canopy-level water-use were measured. Alteration of hydroxycinnamic acid content led to significant decreases in mesophyll cell wall thickness (−14%), and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated increased photosynthesis, resulting in no change in intrinsic water-use efficiency (ratio of photosynthesis/stomatal conductance). The leaf hydraulic conductance was also unchanged; however, the transgenics showed small, but significant increase in above-ground biomass (+12.5%), and canopy-level water-use efficiency (+8.8%; ratio of above-ground biomass/ water-used) and performed better under low water-level. Our results demonstrate that changes in cell wall composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops like rice. However, attempts to improve photosynthetic water-use efficiency will need to enhance mesophyll conductance and photosynthesis whilst maintaining or decreasing stomatal conductance.

Due to the difficulty in accessing to gametes and zygotes in flowering plants, which are controlled in single cells deeply embedded in multiple tissues, little is known about how the initiation of plant embryogenesis may reflect or contrast from such systems in other eukaryotes. In this study we has developed an approach of isolation and transfection of rice egg cells and zygotes for cellular localization of rice cell cycle factors (KRP5, KRP4 and FB3), which opened a pathway to monitor protein expression in rice egg cells and zygotes at different developmental stages. The advantageous feature of isolated rice cells may serve as an ideal system for studying the molecular mechanism underlying the rice zygotic division to initiate seed formation.