Phenotyping Alfalfa (Medicago sativa L.) Root Structure Architecture via Integrating Confident Machine Learning with ResNet-18
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59 BASIC BIOLOGICAL SCIENCES↗
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Unbalancing Symbiotic Nitrogen Fixation: Can We Make Effectiveness More Effective?
One of the most critical uses of carbon fuels is in generating nitrogen fertilizer. Atmospheric dinitrogen is too stable to be used directly by plants, so plants need other chemical forms of nitrogen. Nitrogen fertilizer production uses ~4% of the world’s natural gas. Making N fertilizers leverages methane’s energy content by enabling additional energy to be captured into food and fiber through increased photosynthesis. Manufacturing fertilizer is essential—without it there would only be enough combined nitrogen to feed ~3 billion of the world’s ~7 billion people. How to alter this addiction is not obvious, but the best chance to secure substantial and sustainable amounts of future N might be through symbiotic nitrogen fixation (SNF). SNF describes mutualistic interactions where nitrogen-fixing bacteria provide fixed nitrogen to their plant hosts, freeing them from the need for nitrogen fertilizers. SNF already occurs on a large scale; "biological nitrogen” contributes about 5 Tg N/yr to American agriculture, a quarter of the nitrogen needed. Improving SNF and replacing inorganic fertilizers are both important goals in establishing sustainable and more energy-efficient farming. SNF has been used in agriculture for millennia, largely by using legumes like beans or alfalfa in mixed cropping systems. Legume SNF occurs in root nodules, organs that develop after a growing root is infected by bacteria called rhizobia. In the plant cells of a legume nodule, specialized forms of rhizobia fix nitrogen and can produce enough ammonia to supply the growing plant. The exchange of photosynthetically derived plant carbon compounds for nitrogen reduced by the bacteria is the engine that drives the symbiosis. Important details about the organization of development and metabolism in SNF remain to be determined, such as how plant and bacterial metabolism are coordinated as nitrogen is being fixed and what governs the overall level of nitrogen fixation. Most bacterial mutations that affect SNF completely block fixation (e.g. are Fix – ), providing a limited window into the feedback interactions that must be part of the process. We have investigated an unusual mutant of Sinorhizobium meliloti, a symbiotic partner of alfalfa, that fixes dinitrogen at a normal rate (i.e., it is Fix + ) but is not effective in supporting plant growth (i.e., it is ineffective or Eff – ). We have shown that the mutant has a specific mutation in the glnD gene, which codes for a protein that regulates the bacterial response to nitrogen stress, among other key pathways. A Fix + Eff – phenotype contains an unavoidable puzzle—how is it possible for the bacteria to fix nitrogen at a normal rate and without benefiting the nitrogen starved plant host? After eliminating other possibilities, we proposed that the bacteria synthesize a nitrogen-containing compound that the plant can’t use so that acquiring this compound does not relieve plant nitrogen stress. Extracts of nodules made by glnD mutant strains have high concentrations of pyruvate canaline oxime (PCO), a derivative of the plant defensive compound canaline, a toxic analog of ornithine, an amino acid. Many legumes produce canaline and the related arginine mimic canavanine. For reasons we do not yet understand, the mutation in glnD appears to trigger significant overproduction of canaline in a defense response that leads to substantial synthesis of PCO. We expected the bacteria to be able to use PCO, but we have not been able to grow S. meliloti on PCO under several conditions. PCO thus appears to be a metabolic dead end for both the plant and bacteria. We also examined bacterial catabolic pathways that degrade arginine and canavanine and showed that mutation of these did not alter the symbiosis in an obvious way. In related experiments to examine the metabolic development of root nodules, we carried out an ambitious experiment to simultaneously measure metabolites and protein changes during nodule maturation. For these experiments we used a related symbiotic interaction between S. medicae and Medicago truncatula. M. truncatula has a simpler genome than alfalfa so we could identify plant proteins based on predictions from the DNA sequence. Nodules formed on Medicago grow linearly, with the least mature tissues at the tip. Because development of nitrogen fixation is accompanied by the production of the pigmented plant oxygen carrier protein leghemoglobin, we were able to locate and manually separate the tip, transitional, and mature nitrogen-fixing regions from each other. Advances in proteomic and metabolomic techniques allowed us to analyze these tissues from a pool of as few as 10 nodules. We observed correlated transitions of various enzymes in the plant and bacterial proteomes from those characteristic of free-living metabolism to the microaerobic metabolism of the nitrogen-fixing tissues. The metabolome could not be separated into plant and bacterial domains but was consistent with this overall transition. A subsequent paper examined proteolysis in nodule bacteria. Protein turnover in mature regions of the nodule is significant. We were able to show complexes between various proteins and important proteases, using precipitation capture techniques to isolate the proteases and sensitive proteomic analysis to reveal the associated proteins.
09 BIOMASS FUELS↗
Investigating biological nitrogen fixation via single-cell transcriptomics
The extensive use of nitrogen fertilizers has detrimental environmental consequences, and it is essential for society to explore sustainable alternatives. One promising avenue is engineering root nodule symbiosis, a naturally occurring process in certain plant species within the nitrogen-fixing clade, into non-leguminous crops. Advancements in single-cell transcriptomics provide unprecedented opportunities to dissect the molecular mechanisms underlying root nodule symbiosis at the cellular level. This review summarizes key findings from single-cell studies in Medicago truncatula, Lotus japonicus, and Glycine max. We highlight how these studies address fundamental questions about the development of root nodule symbiosis, including the following findings: (i) single-cell transcriptomics has revealed a conserved transcriptional program in root hair and cortical cells during rhizobial infection, suggesting a common infection pathway across legume species; (ii) characterization of determinate and indeterminate nodules using single-cell technologies supports the compartmentalization of nitrogen fixation, assimilation, and transport into distinct cell populations; (iii) single-cell transcriptomics data have enabled the identification of novel root nodule symbiosis genes and provided new approaches for prioritizing candidate genes for functional characterization; and (iv) trajectory inference and RNA velocity analyses of single-cell transcriptomics data have allowed the reconstruction of cellular lineages and dynamic transcriptional states during root nodule symbiosis.
Lotus japonicus↗
Combining GWAS and population genomic analyses to characterize coevolution in a legume‐rhizobia symbiosis
Abstract The mutualism between legumes and rhizobia is clearly the product of past coevolution. However, the nature of ongoing evolution between these partners is less clear. To characterize the nature of recent coevolution between legumes and rhizobia, we used population genomic analysis to characterize selection on functionally annotated symbiosis genes as well as on symbiosis gene candidates identified through a two‐species association analysis. For the association analysis, we inoculated each of 202 accessions of the legume host Medicago truncatula with a community of 88 Sinorhizobia (Ensifer) meliloti strains. Multistrain inoculation, which better reflects the ecological reality of rhizobial selection in nature than single‐strain inoculation, allows strains to compete for nodulation opportunities and host resources and for hosts to preferentially form nodules and provide resources to some strains. We found extensive host by symbiont, that is, genotype‐by‐genotype, effects on rhizobial fitness and some annotated rhizobial genes bear signatures of recent positive selection. However, neither genes responsible for this variation nor annotated host symbiosis genes are enriched for signatures of either positive or balancing selection. This result suggests that stabilizing selection dominates selection acting on symbiotic traits and that variation in these traits is under mutation‐selection balance. Consistent with the lack of positive selection acting on host genes, we found that among‐host variation in growth was similar whether plants were grown with rhizobia or N‐fertilizer, suggesting that the symbiosis may not be a major driver of variation in plant growth in multistrain contexts.
59 BASIC BIOLOGICAL SCIENCES↗
Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen–fixing clade
Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root–nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.
59 BASIC BIOLOGICAL SCIENCES↗
Rapid changes in agricultural land use and hydrology in the Driftless Region
Annual cropping systems are common in the Driftless Region of the U.S. Midwest, but soil degradation is prone to happen in such systems due to the rugged topography of the region. Recent rapid increases in row crop area have been noted in this region, with annual precipitation and hydrologic extremes on the rise in recent decades. The aim of this research to use geospatial datasets and tools in order to assess the regional trends in land use, precipitation, and hydrologic change and quantify the relationship between these environmental trends. Between 2006 and 2017, substantial row crop expansion of 10,000 ha or more was common across HUC 8 (Hydrologic Unit Code 8) watersheds in our study area. Expansion occurred mainly on steeper slopes, converting existing grasslands or alfalfa (Medicago sativa L.) to row crops. Classifying land as planted (in row crops), plantable (in row crops or could be converted), and unplantable (unable to be converted) revealed that Driftless Region watersheds have ~30–50% of plantable land available for future expansion. Annual precipitation was highly variable during this time period but had a general increasing trend. On average, precipitation showed higher correlation to streamflow compared to row crop expansion across 27 USGS river gage drainage basins in our study area. However, when the increase in row crop area was significant and was accompanied by increasing precipitation, stronger correlation between row crop area and annual streamflow was exhibited. This finding suggests that row crop expansion acts to enhance the effects of increasing precipitation on local hydrology.
54 ENVIRONMENTAL SCIENCES↗
Soil carbon change in intensive agriculture after 25 years of conservation management
Changes in soil organic carbon (SOC) and nitrogen (SON) are strongly affected by land management but few long-term comparative studies have surveyed changes throughout the whole soil profile. We quantified 25-year SOC and SON changes to 1 m in 10 replicate ecosystems at an Upper Midwest, USA site. We compared four annual cropping systems in maize (Zea mays)-soybean (Glycine max)-winter wheat (Triticum aestivum) rotations, each managed differently (Conventional, No-till, Reduced input, and Biologically based); in three managed perennial systems (hybrid Poplar (Populus × euramericana), Alfalfa (Medicago sativa), and Conifer (Pinus spp.); and in three successional systems (Early, Mid- and Late succession undergoing a gradual change in species composition and structure over time). Both Reduced input and Biologically based systems included winter cover crops. Neither SOC nor SON changed significantly in the Conventional or Late successional systems over 25 years. All other systems gained SOC and SON to different degrees. SOC accrual was fastest in the Early successional system (0.8 ± 0.1 Mg C ha –1 y –1 ) followed by Alfalfa and Conifer (avg. 0.7 ± 0.1 Mg C ha –1 y –1 ), Poplar, Reduced input, and Biologically based systems (avg. 0.4 ± 0.1 Mg C ha –1 y –1 ), and Mid-successional and No-till systems (0.3 and 0.2 Mg C ha –1 y –1 , respectively). Over the most recent 12 years, rates of SOC accrual slowed in all systems except Reduced input and Mid-successional. There was no evidence of SOC loss at depth in any system, including No-till. Rates of SON accrual ranged from 64.7 to 0.8 kg N ha –1 y –1 in the order Alfalfa ≥ Early successional > Reduced input and Biologically based ≥ Poplar > No-till and Conifer > Mid-successional systems. Pyrogenic C levels in the Conventional, Early, and Late successional systems were similar despite 17 years of annual burning in the Early successional system (~ 15 % of SOC to 50 cm, on average, and ~40 % of SOC from 50 to 100 cm). Results underscore the importance of cover crops, perennial crops, and no-till options for sequestering whole profile C in intensively managed croplands.
60 APPLIED LIFE SCIENCES↗
Insights into the substrate specificity, structure, and dynamics of plant histidinol-phosphate aminotransferase (HISN6)
Histidinol-phosphate aminotransferase is the sixth protein (hence HISN6) in the histidine biosynthetic pathway in plants. HISN6 is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of imidazole acetol phosphate into L-histidinol phosphate (HOLP). Here, we show that plant HISN6 enzymes are closely related to the orthologs from Chloroflexota. The studied example, HISN6 from Medicago truncatula (MtHISN6), exhibits a surprisingly high affinity for HOLP, which is much higher than reported for bacterial homologs. Moreover, unlike the latter, MtHISN6 does not transaminate phenylalanine. High-resolution crystal structures of MtHISN6 in the open and closed states, as well as the complex with HOLP and the apo structure without PLP, bring new insights into the enzyme dynamics, pointing at a particular role of a string-like fragment that oscillates near the active site and participates in the HOLP binding. When MtHISN6 is compared to bacterial orthologs with known structures, significant differences arise in or near the string region. The high affinity of MtHISN6 appears linked to the particularly tight active site cavity. Finally, a virtual screening against a library of over 1.3 mln compounds revealed three sites in the MtHISN6 structure with the potential to bind small molecules. Such compounds could be developed into herbicides inhibiting plant HISN6 enzymes absent in animals, which makes them a potential target for weed control agents.
59 BASIC BIOLOGICAL SCIENCES↗
The core metabolome and root exudation dynamics of three phylogenetically distinct plant species
Abstract Root exudates are plant-derived, exported metabolites likely shaping root-associated microbiomes by acting as nutrients and signals. However, root exudation dynamics are unclear and thus also, if changes in exudation are reflected in changes in microbiome structure. Here, we assess commonalities and differences between exudates of different plant species, diurnal exudation dynamics, as well as the accompanying methodological aspects of exudate sampling. We find that exudates should be collected for hours rather than days as many metabolite abundances saturate over time. Plant growth in sterile, nonsterile, or sugar-supplemented environments significantly alters exudate profiles. A comparison of Arabidopsis thaliana , Brachypodium distachyon , and Medicago truncatula shoot, root, and root exudate metabolite profiles reveals clear differences between these species, but also a core metabolome for tissues and exudates. Exudate profiles also exhibit a diurnal signature. These findings add to the methodological and conceptual groundwork for future exudate studies to improve understanding of plant-microbe interactions.
59 BASIC BIOLOGICAL SCIENCES↗
Spatial co-transcriptomics reveals discrete stages of the arbuscular mycorrhizal symbiosis
Abstract The symbiotic interaction of plants with arbuscular mycorrhizal (AM) fungi is ancient and widespread. Plants provide AM fungi with carbon in exchange for nutrients and water, making this interaction a prime target for crop improvement. However, plant–fungal interactions are restricted to a small subset of root cells, precluding the application of most conventional functional genomic techniques to study the molecular bases of these interactions. Here we used single-nucleus and spatial RNA sequencing to explore both Medicago truncatula and Rhizophagus irregularis transcriptomes in AM symbiosis at cellular and spatial resolution. Integrated, spatially registered single-cell maps revealed infected and uninfected plant root cell types. We observed that cortex cells exhibit distinct transcriptome profiles during different stages of colonization by AM fungi, indicating dynamic interplay between both organisms during establishment of the cellular interface enabling successful symbiosis. Our study provides insight into a symbiotic relationship of major agricultural and environmental importance and demonstrates a paradigm combining single-cell and spatial transcriptomics for the analysis of complex organismal interactions.
59 BASIC BIOLOGICAL SCIENCES↗
Netting and pan traps fail to identify the pollinator guild of an agricultural crop
Pollinator decline is expected to cause significant reductions in food production and plant reproduction. Quantifying the impact of pollinator decline on food production requires survey methods that can identify insect and bee species responsible for pollination of specific crops. To address this issue, we compared the effectiveness of two survey methods, netting and pan traps, at capturing the pollinators of alfalfa, Medicago sativa. Alfalfa is a major component of forage for cows and an important ingredient in chicken feed. We also examined bee species richness and diversity with these two survey methods, and compared these measures among three different colors of pan traps. Netting was more effective at capturing known pollinators of alfalfa, especially those belonging to the Bombus and Apis genera. Pan traps captured a higher bee diversity relative to netting and, like previous studies, each survey method and each trap color was more efficient at capturing certain bee genera. However, without a priori knowledge of pollinators, neither survey method could identify which of the bee species captured could pollinate alfalfa. We therefore recommend direct observations when the goal of a study is to identify pollinators or link pollinator decline to food production.
54 ENVIRONMENTAL SCIENCES↗
Structural and mechanistic insights into the bifunctional HISN2 enzyme catalyzing the second and third steps of histidine biosynthesis in plants
The second and third steps of the histidine biosynthetic pathway (HBP) in plants are catalyzed by a bifunctional enzyme–HISN2. The enzyme consists of two distinct domains, active respectively as a phosphoribosyl-AMP cyclohydrolase (PRA-CH) and phosphoribosyl-ATP pyrophosphatase (PRA-PH). The domains are analogous to single-domain enzymes encoded by bacterial hisI and hisE genes, respectively. The calculated sequence similarity networks between HISN2 analogs from prokaryotes and eukaryotes suggest that the plant enzymes are closest relatives of those in the class of Deltaproteobacteria. In this work, we obtained crystal structures of HISN2 enzyme from Medicago truncatula (MtHISN2) and described its architecture and interactions with AMP. The AMP molecule bound to the PRA-PH domain shows positioning of the N1-phosphoribosyl relevant to catalysis. AMP bound to the PRA-CH domain mimics a part of the substrate, giving insights into the reaction mechanism. The latter interaction also arises as a possible second-tier regulatory mechanism of the HBP flux, as indicated by inhibition assays and isothermal titration calorimetry.
59 BASIC BIOLOGICAL SCIENCES↗
Antifungal symbiotic peptide NCR044 exhibits unique structure and multifaceted mechanisms of action that confer plant protection
In the indeterminate nodules of a model legume Medicago truncatula, ~700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel β-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea. In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.
59 BASIC BIOLOGICAL SCIENCES↗
Convergent evolution of NFP -facilitated root nodule symbiosis
The origin and phylogenetic distribution of symbiotic associations between nodulating angiosperms and nitrogen-fixing bacteria have long intrigued biologists. Recent comparative evolutionary analyses have yielded alternative hypotheses: a multistep pathway of independent gains and losses of root nodule symbiosis vs. a single gain followed by numerous losses. A detailed reconstruction of the history of genes involved in signaling between nitrogen-fixing bacteria and potential hosts, particularly lipo-chitooligosaccharide (LCO) signaling, is needed to distinguish between these hypotheses. LCO recognition by plants involves the Nod Factor Perception ( NFP ) gene family; in the legume model Medicago truncatula (Fabales), MtNFP is essential for establishing rhizobial symbiosis. Here, we document convergent evolution of NFP , indicating multiple origins of LCO-driven symbiosis. In contrast to previous models that explain the recruitment of NFP via a single duplication in the ancestor of the nitrogen-fixing clade, our phylogenomic and synteny results suggest this duplication does not span the entire clade. Tandem duplication in a common ancestor of Cucurbitales and Rosales resulted in the NFP1 and NFP2 groups. In contrast, the phylogenetically closest paralog of MtNFP is MtLYR1 , located on a different chromosome within a large syntenic block. All available data indicate that a large-scale duplication resulted in MtNFP and MtLYR1 , likely corresponding to a whole-genome duplication in an ancestor of subfamily Papilionoideae of Fabaceae. We show that MtNFP and the NFP2 -like group are not orthologous, indicating multiple independent gains of NFP -based LCO signaling. This molecular convergence provides a possible mechanism for multiple gains of root nodule symbiosis across the nitrogen-fixing clade.
LCO signaling↗
Root and xylem anatomy varies with root length, root order, soil depth and environment in intermediate wheatgrass (Kernza®) and alfalfa
Abstract Background and Aims Deep roots (i.e. >1 m depth) are important for crops to access water when the topsoil is dry. Root anatomy and hydraulic conductance play important roles in the uptake of soil water, particularly water located deep in the soil. We investigated whether root and xylem anatomy vary as a function of root type, order and length, or with soil depth in roots of two deep-rooted perennial crops: intermediate wheatgrass [Thinopyrum intermedium (Kernza®)] and alfalfa (Medicago sativa). We linked the expression of these anatomical traits to the plants’ capacity to take up water from deep soil layers. Methods Using laser ablation tomography, we compared the roots of the two crops for cortical area, number and size of metaxylem vessels, and their estimated root axial hydraulic conductance (ERAHCe). The deepest roots investigated were located at soil depths of 2.25 and at 3.5 m in the field and in rhizoboxes, respectively. Anatomical differences were characterized along 1-m-long individual roots, among root types and orders, as well as between environmental conditions. Key Results For both crops, a decrease in the number and diameter, or both, of metaxylem vessels along individual root segments and with soil depth in the field resulted in a decrease in ERAHCe. Alfalfa, with a greater number of metaxylem vessels per root throughout the soil profile and, on average, a 4-fold greater ERAHCe, took up more water from the deep soil layers than intermediate wheatgrass. Root anatomical traits were significantly different across root types, classes and growth conditions. Conclusions Root anatomical traits are important tools for the selection of crops with enhanced exploitation of deep soil water. The development and breeding of perennial crops for improved subsoil exploitation will be aided by greater understanding of root phenotypes linked to deep root growth and activity.
Plant Sciences↗
UDP-glucuronic acid decarboxylase in alfalfa: a target to improve ruminal digestibility of stems
Alfalfa (Medicago sativa) has a high nutritional value, but poor digestibility of the stems limits its value as an energy source in ruminant diets. Xylan and lignin negatively affect cell wall digestibility, whereas pectins have high digestibility in the rumen. In plants, UDP-xylose synthase (UXS) catalyses the decarboxylation of UDP-glucuronic acid to form UDP-xylose in an irreversible step that is key for xylan synthesis. Here, we functionally characterized two UXS genes in alfalfa, namely MsaUXS2 and MsaUXS4, and investigated their impact on ruminal digestibility. Both genes are more highly expressed in stems than leaves, and the enzymes have UDP-glucuronic acid decarboxylase activity in vitro. Silencing of MsaUXS2 and MsaUXS4 via RNAi altered plant growth and resulted in a 40% decrease in xylose, a 115% increase in arabinose, and a 60% increase in galacturonic acid in the polysaccharide matrix as well as a 20% decrease in lignin in the cell wall. Together, our results show a major role for UXS2 and UXS4 in xylan synthesis and secondary cell wall deposition in alfalfa. Additionally, in vitro rumen digestibility assays for the silenced lines had on average 30% increased gas production at 24 h, demonstrating the potential of targeting UXS genes to increase stem digestibility.
UDP-xylose synthase↗
Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca 2+ Responsiveness
Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca 2+ -controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca 2+ -dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca 2+ -binding motifs. Here we selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca 2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast ( Saccharomyces cerevisiae ) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca 2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca 2+ -controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca 2+ -dependent contractility.
59 BASIC BIOLOGICAL SCIENCES↗
Major facilitator family transporters specifically enhance caffeyl alcohol uptake during C‐lignin biosynthesis
The mode of transport of lignin monomers to the sites of polymerization in the apoplast remains controversial. C-Lignin is a recently discovered form of lignin found in some seed coats that is composed exclusively of units derived from caffeyl alcohol. RNA-seq and proteome analyses identified a number of transporters co-expressed with C-lignin deposition in the seed coat of Cleome hassleriana. Cloning and influx/efflux analysis assays in yeast identified two low-affinity transporters, ChPLT3 and ChSUC1, that were active with caffeyl alcohol but not with the classical monolignols p-coumaryl, coniferyl, and sinapyl alcohols, consistent with molecular modeling and docking studies. Expression of ChPLT3 in Arabidopsis seedlings enhanced root growth in the presence of caffeyl alcohol, and expression of ChPLT3 and ChSUC1 correlated with lignin C-unit content in hairy roots of Medicago truncatula. We present a model, consistent with phylogenetic and evolutionary considerations, whereby passive caffeyl alcohol transport may be supplemented by hitchhiking on secondary active transporters to ensure the synthesis of C-lignin, and inhibition of synthesis of G-lignin, in the apoplast.
59 BASIC BIOLOGICAL SCIENCES↗