Fifteen compelling open questions in plant cell biology.

As scientists, we are at least as excited about the open questions-the things we do not know-as the discoveries. Here, we asked 15 experts to describe the most compelling open questions in plant cell biology. These are their questions: How are organelle identity, domains, and boundaries maintained under the continuous flux of vesicle trafficking and membrane remodeling? Is the plant cortical microtubule cytoskeleton a mechanosensory apparatus? How are the cellular pathways of cell wall synthesis, assembly, modification, and integrity sensing linked in plants? Why do plasmodesmata open and close? Is there retrograde signaling from vacuoles to the nucleus? How do root cells accommodate fungal endosymbionts? What is the role of cell edges in plant morphogenesis? How is the cell division site determined? What are the emergent effects of polyploidy on the biology of the cell, and how are any such "rules" conditioned by cell type? Can mechanical forces trigger new cell fates in plants? How does a single differentiated somatic cell reprogram and gain pluripotency? How does polarity develop de-novo in isolated plant cells? What is the spectrum of cellular functions for membraneless organelles and intrinsically disordered proteins? How do plants deal with internal noise? How does order emerge in cells and propagate to organs and organisms from complex dynamical processes? We hope you find the discussions of these questions thought provoking and inspiring.

Information theory and machine learning illuminate large-scale metabolomic responses of Brachypodium distachyon to environmental change.

Plant responses to environmental change are mediated via changes in cellular metabolomes. However, <5% of signals obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) can be identified, limiting our understanding of how metabolomes change under biotic/abiotic stress. To address this challenge, we performed untargeted LC-MS/MS of leaves, roots, and other organs of Brachypodium distachyon (Poaceae) under 17 organ-condition combinations, including copper deficiency, heat stress, low phosphate, and arbuscular mycorrhizal symbiosis. We found that both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We found that 1 week of copper deficiency shielded the root, but not the leaf metabolome, from perturbation due to heat stress. Machine learning (ML)-based analysis annotated approximately 81% of the fragmented peaks versus approximately 6% using spectral matches alone. We performed one of the most extensive validations of ML-based peak annotations in plants using thousands of authentic standards, and analyzed approximately 37% of the annotated peaks based on these assessments. Analyzing responsiveness of each predicted metabolite class to environmental change revealed significant perturbations of glycerophospholipids, sphingolipids, and flavonoids. Co-accumulation analysis further identified condition-specific biomarkers. To make these results accessible, we developed a visualization platform on the Bio-Analytic Resource for Plant Biology website (https://bar.utoronto.ca/efp_brachypodium_metabolites/cgi-bin/efpWeb.cgi), where perturbed metabolite classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and stress adaptation.

KIN3 impacts arbuscular mycorrhizal symbiosis and promotes fungal colonisation in Medicago truncatula.

Arbuscular mycorrhizal fungi help their host plant in the acquisition of nutrients, and this association is itself impacted by soil nutrient levels. High phosphorus levels inhibit the symbiosis, whereas high nitrogen levels enhance it. The genetic mechanisms regulating the symbiosis in response to soil nutrients are poorly understood. Here, we characterised the symbiotic phenotypes in four Medicago truncatula Tnt1-insertion mutants affected in arbuscular mycorrhizal colonisation. We located their Tnt1 insertions and identified alleles for two genes known to be involved in mycorrhization, RAM1 and KIN3. We compared the effects of the kin3-2 and ram1-4 mutations on gene expression, revealing that the two genes alter the expression of overlapping but not identical gene sets, suggesting that RAM1 acts upstream of KIN3. Additionally, KIN3 appears to be involved in the suppression of plant defences in response to the fungal symbiont. KIN3 is located on the endoplasmic reticulum of arbuscule-containing cortical cells, and kin3-2 mutants plants hosted significantly fewer arbuscules than the wild type. KIN3 plays an essential role in the symbiotic response to soil nitrogen levels, as, contrary to wild-type plants, the kin3-2 mutant did not exhibit increased root colonisation under high nitrogen.

A genetically encoded biosensor reveals spatiotemporal variation in cellular phosphate content in Brachypodium distachyon mycorrhizal roots.

Arbuscular mycorrhizal (AM) symbiosis is accompanied by alterations to root cell metabolism and physiology, and to the pathways of orthophosphate (Pi) entry into the root, which increase with Pi delivery to cortical cells via arbuscules. How AM symbiosis influences the Pi content and Pi response dynamics of cells in the root cortex and epidermis is unknown. Using fluorescence resonance energy transfer (FRET)-based Pi biosensors, we mapped the relative cytosolic and plastidic Pi content of Brachypodium distachyon mycorrhizal root cells, analyzed responses to extracellular Pi and traced extraradical hyphae-mediated Pi transfer to colonized cells. Colonized cortical cells had a higher cytosolic Pi content relative to noncolonized cortical and epidermal cells, while plastidic Pi content was highest in cells at the infection front. Pi application to the entire mycorrhizal root resulted in transient changes in cytosolic Pi that differed in direction and magnitude depending on cell type and arbuscule status; cells with mature arbuscules showed a substantial transient increase in cytosolic Pi while those with collapsed arbuscules showed a decrease. Directed Pi application to extraradical hyphae resulted in measurable changes in cytosolic Pi of colonized cells 18 h after application. Our experiments reveal that cells within a mycorrhizal root vary in Pi content and Pi response dynamics.

Transcriptomic analysis of field-droughted sorghum from seedling to maturity reveals biotic and metabolic responses.

Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [Sorghum bicolor (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants' roots.

Constitutive Overexpression of RAM1 Leads to an Increase in Arbuscule Density in Brachypodium distachyon.

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial association of plants and fungi of the subphylum Glomeromycotina. Endosymbiotic AM fungi colonize the inner cortical cells of the roots, where they form branched hyphae called arbuscules that function in nutrient exchange with the plant. To support arbuscule development and subsequent bidirectional nutrient exchange, the root cortical cells undergo substantial transcriptional reprogramming. REDUCED ARBUSCULAR MYCORRHIZA1 (RAM1), previously studied in several dicot plant species, is a major regulator of this cortical cell transcriptional program. Here, we generated ram1 mutants and RAM1 overexpressors in a monocot, Brachypodium distachyon. The AM phenotypes of two ram1 lines revealed that RAM1 is only partly required to enable arbuscule development in B. distachyon Transgenic lines constitutively overexpressing BdRAM1 showed constitutive expression of AM-inducible genes even in the shoots. Following inoculation with AM fungi, BdRAM1-overexpressing plants showed higher arbuscule densities relative to controls, indicating the potential to manipulate the relative proportion of symbiotic interfaces via modulation of RAM1 However, the overexpressors also show altered expression of hormone biosynthesis genes and aberrant growth patterns, including stunted bushy shoots and poor seed set. While these phenotypes possibly provide additional clues about the scope of influence of BdRAM1, they also indicate that directed approaches to increase the density of symbiotic interfaces will require a more focused, potentially cell type specific manipulation of transcription factor gene expression.

Plant Signaling and Metabolic Pathways Enabling Arbuscular Mycorrhizal Symbiosis.

Plants have lived in close association with arbuscular mycorrhizal (AM) fungi for over 400 million years. Today, this endosymbiosis occurs broadly in the plant kingdom where it has a pronounced impact on plant mineral nutrition. The symbiosis develops deep within the root cortex with minimal alterations in the external appearance of the colonized root; however, the absence of macroscopic alterations belies the extensive signaling, cellular remodeling, and metabolic alterations that occur to enable accommodation of the fungal endosymbiont. Recent research has revealed the involvement of a novel N-acetyl glucosamine transporter and an alpha/beta-fold hydrolase receptor at the earliest stages of AM symbiosis. Calcium channels required for symbiosis signaling have been identified, and connections between the symbiosis signaling pathway and key transcriptional regulators that direct AM-specific gene expression have been established. Phylogenomics has revealed the existence of genes conserved for AM symbiosis, providing clues as to how plant cells fine-tune their biology to enable symbiosis, and an exciting coalescence of genome mining, lipid profiling, and tracer studies collectively has led to the conclusion that AM fungi are fatty acid auxotrophs and that plants provide their fungal endosymbionts with fatty acids. Here, we provide an overview of the molecular program for AM symbiosis and discuss these recent advances.

A Phosphate-Dependent Requirement for Transcription Factors IPD3 and IPD3L During Arbuscular Mycorrhizal Symbiosis in Medicago truncatula.

During arbuscular mycorrhizal (AM) symbiosis, activation of a symbiosis signaling pathway induces gene expression necessary for accommodation of AM fungi. Here, we focus on pathway components Medicago truncatula INTERACTING PROTEIN OF DOES NOT MAKE INFECTIONS 3 (IPD3) and IPD3 LIKE (IPD3L), which are potential orthologs of Lotus japonicus CYCLOPS, a transcriptional regulator essential for AM symbiosis. In the double mutant ipd3 ipd3l, hyphal entry through the epidermis and overall colonization levels are reduced relative to the wild type but fully developed arbuscules are present in the cortex. In comparison with the wild type, colonization of ipd3 ipd3l is acutely sensitive to higher phosphate levels in the growth medium, with a disproportionate decrease in epidermal penetration, overall colonization, and symbiotic gene expression. When constitutively expressed in ipd3 ipd3l, an autoactive DOES NOT MAKE INFECTIONS 3 induces the expression of transcriptional regulators REDUCED ARBUSCULAR MYCORRHIZA 1 and REQUIRED for ARBUSCULE DEVELOPMENT 1, providing a possible avenue for arbuscule development in the absence of IPD3 and IPD3L. An increased sensitivity of ipd3 ipd3l to GA3 suggests an involvement of DELLA. The data reveal partial redundancy in the symbiosis signaling pathway, which may ensure robust signaling in low-phosphorus environments, while IPD3 and IPD3L maintain signaling in higher-phosphorus environments. The latter may buffer the pathway from short-term variation in phosphorus levels encountered by roots during growth in heterogeneous soil environments.

Accumulation of phosphoinositides in distinct regions of the periarbuscular membrane.

Phosphoinositides and phosphatidic acid are small anionic lipids that comprise a minor proportion of total membrane lipids in eukaryotic cells but influence a broad range of cellular processes including endomembrane trafficking, signaling, exocytosis and endocytosis. To investigate the spatial distribution of phosphoinositides during arbuscular mycorrhizal symbiosis, we generated fluorescent reporters of PI(4,5)P2 and PI4P, as well as phosphatidic acid and diacylglycerol and used them to monitor lipid distribution on the cytoplasmic side of membrane bilayers in colonized cortical cells. The PI4P reporter accumulated strongly on the periarbuscular membrane (PAM) and transiently labeled Golgi bodies, while the PA reporter showed differential labeling of endomembranes and the PAM. Surprisingly, the PI(4,5)P2 reporter accumulated in small, discrete regions of the PAM on the arbuscule trunks, frequently in two regions on opposing sides of the hypha. A mutant reporter with reduced PI(4,5)P2 binding capacity did not show these accumulations. The PI(4,5)P2 -rich regions were detected at all phases of arbuscule development following branching, co-localized with membrane marker proteins potentially indicating high membrane bilayer content, and were associated with an alteration in morphology of the hypha. A possible analogy to the biotrophic interfacial membrane complex formed in rice infected with Magnaporthe orzyae is discussed.

Genome and evolution of the arbuscular mycorrhizal fungus Diversispora epigaea (formerly Glomus versiforme) and its bacterial endosymbionts.

Arbuscular mycorrhizal (AM) fungi form endosymbioses with most plants, and they themselves are hosts for Mollicutes/Mycoplasma-related endobacteria (MRE). Despite their significance, genomic information for AM fungi and their MRE are relatively sparse, which hinders our understanding of their biology and evolution. We assembled the genomes of the AM fungus Diversispora epigaea (formerly Glomus versiforme) and its MRE and performed comparative genomics and evolutionary analyses. The D. epigaea genome showed a pattern of substantial gene duplication and differential evolution of gene families, including glycosyltransferase family 25, whose activities are exclusively lipopolysaccharide biosynthesis. Genes acquired by horizontal transfer from bacteria possibly function in defense against foreign DNA or viruses. The MRE population was diverse, with multiple genomes displaying characteristics of differential evolution and encoding many MRE-specific genes as well as genes of AM fungal origin. Gene family expansion in D. epigaea may enhance adaptation to both external and internal environments, such as expansion of kinases for signal transduction upon external stimuli and expansion of nucleoside salvage pathway genes potentially for competition with MRE, whose genomes lack purine and pyrimidine biosynthetic pathways. Collectively, this metagenome provides high-quality references and begins to reveal the diversity within AM fungi and their MRE.

Diverse Sorghum bicolor accessions show marked variation in growth and transcriptional responses to arbuscular mycorrhizal fungi.

Sorghum is an important crop grown worldwide for feed and fibre. Like most plants, it has the capacity to benefit from symbioses with arbuscular mycorrhizal (AM) fungi, and its diverse genotypes likely vary in their responses. Currently, the genetic basis of mycorrhiza-responsiveness is largely unknown. Here, we investigated transcriptional and physiological responses of sorghum accessions, founders of a bioenergy nested association mapping panel, for their responses to four species of AM fungi. Transcriptome comparisons across four accessions identified mycorrhiza-inducible genes; stringent filtering criteria revealed 278 genes that show mycorrhiza-inducible expression independent of genotype and 55 genes whose expression varies with genotype. The latter suggests variation in phosphate transport and defence across these accessions. The mycorrhiza growth and nutrient responses of 18 sorghum accessions varied tremendously, ranging from mycorrhiza-dependent to negatively mycorrhiza-responsive. Additionally, accessions varied in the number of AM fungi to which they showed positive responses, from one to several fungal species. Mycorrhiza growth and phosphorus responses were positively correlated, whereas expression of two mycorrhiza-inducible phosphate transporters, SbPT8 and SbPT9, correlated negatively with mycorrhizal growth responses. AM fungi improve growth and mineral nutrition of sorghum, and the substantial variation between lines provides the potential to map loci influencing mycorrhiza responses.

Using membrane transporters to improve crops for sustainable food production.

With the global population predicted to grow by at least 25 per cent by 2050, the need for sustainable production of nutritious foods is critical for human and environmental health. Recent advances show that specialized plant membrane transporters can be used to enhance yields of staple crops, increase nutrient content and increase resistance to key stresses, including salinity, pathogens and aluminium toxicity, which in turn could expand available arable land.

Suppression of Arbuscule Degeneration in Medicago truncatula phosphate transporter4 Mutants is Dependent on the Ammonium Transporter 2 Family Protein AMT2;3.

During arbuscular mycorrhizal (AM) symbiosis, the plant gains access to phosphate (Pi) and nitrogen delivered by its fungal symbiont. Transfer of mineral nutrients occurs at the interface between branched hyphae called arbuscules and root cortical cells. In Medicago truncatula, a Pi transporter, PT4, is required for symbiotic Pi transport, and in pt4, symbiotic Pi transport fails, arbuscules degenerate prematurely, and the symbiosis is not maintained. Premature arbuscule degeneration (PAD) is suppressed when pt4 mutants are nitrogen-deprived, possibly the result of compensation by PT8, a second AM-induced Pi transporter. However, PAD is also suppressed in nitrogen-starved pt4 pt8 double mutants, negating this hypothesis and furthermore indicating that in this condition, neither of these symbiotic Pi transporters is required for symbiosis. In M. truncatula, three AMT2 family ammonium transporters are induced during AM symbiosis. To test the hypothesis that suppression of PAD involves AMT2 transporters, we analyzed double and triple Pi and ammonium transporter mutants. ATM2;3 but not AMT2;4 was required for suppression of PAD in pt4, while AMT2;4, but not AMT2;3, complemented growth of a yeast ammonium transporter mutant. In summary, arbuscule life span is influenced by PT4 and ATM2;3, and their relative importance varies with the nitrogen status of the plant.

How grow-and-switch gravitropism generates root coiling and root waving growth responses in Medicago truncatula.

Experimental studies show that plant root morphologies can vary widely from straight gravity-aligned primary roots to fractal-like root architectures. However, the opaqueness of soil makes it difficult to observe how environmental factors modulate these patterns. Here, we combine a transparent hydrogel growth medium with a custom built 3D laser scanner to directly image the morphology of Medicago truncatula primary roots. In our experiments, root growth is obstructed by an inclined plane in the growth medium. As the tilt of this rigid barrier is varied, we find Medicago transitions between randomly directed root coiling, sinusoidal root waving, and normal gravity-aligned morphologies. Although these root phenotypes appear morphologically distinct, our analysis demonstrates the divisions are less well defined, and instead, can be viewed as a 2D biased random walk that seeks the path of steepest decent along the inclined plane. Features of this growth response are remarkably similar to the widely known run-and-tumble chemotactic behavior of Escherichia coli bacteria, where biased random walks are used as optimal strategies for nutrient uptake.

A comprehensive draft genome sequence for lupin (Lupinus angustifolius), an emerging health food: insights into plant-microbe interactions and legume evolution.

Lupins are important grain legume crops that form a critical part of sustainable farming systems, reducing fertilizer use and providing disease breaks. It has a basal phylogenetic position relative to other crop and model legumes and a high speciation rate. Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is gaining popularity as a health food, which is high in protein and dietary fibre but low in starch and gluten-free. We report the draft genome assembly (609 Mb) of NLL cultivar Tanjil, which has captured >98% of the gene content, sequences of additional lines and a dense genetic map. Lupins are unique among legumes and differ from most other land plants in that they do not form mycorrhizal associations. Remarkably, we find that NLL has lost all mycorrhiza-specific genes, but has retained genes commonly required for mycorrhization and nodulation. In addition, the genome also provided candidate genes for key disease resistance and domestication traits. We also find evidence of a whole-genome triplication at around 25 million years ago in the genistoid lineage leading to Lupinus. Our results will support detailed studies of legume evolution and accelerate lupin breeding programmes.

Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza.

During arbuscular mycorrhizal symbiosis (AMS), considerable amounts of lipids are generated, modified and moved within the cell to accommodate the fungus in the root, and it has also been suggested that lipids are delivered to the fungus. To determine the mechanisms by which root cells redirect lipid biosynthesis during AMS we analyzed the roles of two lipid biosynthetic enzymes (FatM and RAM2) and an ABC transporter (STR) that are required for symbiosis and conserved uniquely in plants that engage in AMS. Complementation analyses indicated that the biochemical function of FatM overlaps with that of other Fat thioesterases, in particular FatB. The essential role of FatM in AMS was a consequence of timing and magnitude of its expression. Lipid profiles of fatm and ram2 suggested that FatM increases the outflow of 16:0 fatty acids from the plastid, for subsequent use by RAM2 to produce 16:0 ß-monoacylglycerol. Thus, during AMS, high-level, specific expression of key lipid biosynthetic enzymes located in the plastid and the endoplasmic reticulum enables the root cell to fine-tune lipid biosynthesis to increase the production of ß-monoacylglycerols. We propose a model in which ß-monoacylglycerols, or a derivative thereof, are exported out of the root cell across the periarbuscular membrane for ultimate use by the fungus.

Hyphal Branching during Arbuscule Development Requires Reduced Arbuscular Mycorrhiza1.

During arbuscular mycorrhizal symbiosis, arbuscule development in the root cortical cell and simultaneous deposition of the plant periarbuscular membrane generate the interface for symbiotic nutrient exchange. The transcriptional changes that accompany arbuscule development are extensive and well documented. By contrast, the transcriptional regulators that control these programs are largely unknown. Here, we provide a detailed characterization of an insertion allele of Medicago truncatula Reduced Arbuscular Mycorrhiza1 (RAM1), ram1-3, which reveals that RAM1 is not necessary to enable hyphopodium formation or hyphal entry into the root but is essential to support arbuscule branching. In ram1-3, arbuscules consist only of the arbuscule trunk and in some cases, a few initial thick hyphal branches. ram1-3 is also insensitive to phosphate-mediated regulation of the symbiosis. Transcript analysis of ram1-3 and ectopic expression of RAM1 indicate that RAM1 regulates expression of EXO70I and Stunted Arbuscule, two genes whose loss of function impacts arbuscule branching. Furthermore, RAM1 regulates expression of a transcription factor Required for Arbuscule Development (RAD1). RAD1 is also required for arbuscular mycorrhizal symbiosis, and rad1 mutants show reduced colonization. RAM1 itself is induced in colonized root cortical cells, and expression of RAM1 and RAD1 is modulated by DELLAs. Thus, the data suggest that DELLAs regulate arbuscule development through modulation of RAM1 and RAD1 and that the precise transcriptional control essential to place proteins in the periarbuscular membrane is controlled, at least in part, by RAM1.

DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis.

Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula.

Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans-Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis-specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub-cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens-shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.