ER body-resident myrosinases and tryptophan specialized metabolism modulate root microbiota assembly.

Endoplasmic reticulum (ER) bodies are ER-derived structures that contain a large amount of PYK10 myrosinase, which hydrolyzes tryptophan (Trp)-derived indole glucosinolates (IGs). Given the well-described role of IGs in root-microbe interactions, we hypothesized that ER bodies in roots are important for interaction with soil-borne microbes at the root-soil interface. We used mutants impaired in ER bodies (nai1), ER body-resident myrosinases (pyk10bglu21), IG biosynthesis (myb34/51/122), and Trp specialized metabolism (cyp79b2b3) to profile their root microbiota community in natural soil, evaluate the impact of axenically collected root exudates on soil or synthetic microbial communities, and test their response to fungal endophytes in a mono-association setup. Tested mutants exhibited altered bacterial and fungal communities in rhizoplane and endosphere, respectively. Natural soils and bacterial synthetic communities treated with mutant root exudates exhibited distinctive microbial profiles from those treated with wild-type (WT) exudates. Most tested endophytes severely restricted the growth of cyp79b2b3, a part of which also impaired the growth of pyk10bglu21. Our results suggest that root ER bodies and their resident myrosinases modulate the profile of root-secreted metabolites and thereby influence root-microbiota interactions.

Endoplasmic Reticulum Bodies in the Lateral Root Cap Are Involved in the Direct Transport of Beta-Glucosidase to Vacuoles.

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the ß-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport ß-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders.

MYC transcription factors coordinate tryptophan-dependent defence responses and compromise seed yield in Arabidopsis.

Robust plant immunity negatively affects other fitness traits, including growth and seed production. Jasmonate (JA) confers broad-spectrum protection against plant consumers by stimulating the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins, which in turn relieves repression on transcription factors (TFs) coincident with reduced growth and fecundity. The molecular mechanisms underlying JA-mediated decreases in fitness remain largely unknown. To assess the contribution of MYC TFs to growth and reproductive fitness at high levels of defence, we mutated three MYC genes in a JAZ-deficient mutant (jazD) of Arabidopsis thaliana that exhibits strong defence and low seed yield. Genetic epistasis analysis showed that de-repression of MYC TFs in jazD not only conferred strong resistance to insect herbivory but also reduced shoot and root growth, fruit size and seed yield. We also provided evidence that the JAZ-MYC module coordinates the supply of tryptophan with the production of indole glucosinolates and the proliferation of endoplasmic reticulum bodies that metabolise glucosinolates through the action of ß-glucosidases. Our results establish MYCs as major regulators of growth- and reproductive-defence trade-offs and further indicate that these factors coordinate tryptophan availability with the production of amino acid-derived defence compounds.

The Cellular and Subcellular Organization of the Glucosinolate-Myrosinase System against Herbivores and Pathogens.

Glucosinolates are an important class of secondary metabolites in Brassicales plants with a critical role in chemical defense. Glucosinolates are chemically inactive but can be hydrolyzed by myrosinases to produce a range of chemically active compounds toxic to herbivores and pathogens, thereby constituting the glucosinolate-myrosinase defense system or the mustard oil bomb. During the evolution, Brassicales plants have developed not only complex biosynthetic pathways for production of a large number of glucosinolate structures but also different classes of myrosinases that differ in catalytic mechanisms and substrate specificity. Studies over the past several decades have made important progress in the understanding of the cellular and subcellular organization of the glucosinolate-myrosinase system for rapid and timely detonation of the mustard oil bomb upon tissue damage after herbivore feeding and pathogen infection. Progress has also been made in understanding the mechanisms that herbivores and pathogens have evolved to counter the mustard oil bomb. In this review, we summarize our current understanding of the function and organization of the glucosinolate-myrosinase system in Brassicales plants and discuss both the progresses and future challenges in addressing this complex defense system as an excellent model for analyzing plant chemical defense.

Specialized endoplasmic reticulum-derived vesicles in plants: Functional diversity, evolution, and biotechnological exploitation.

A central role of the endoplasmic reticulum (ER) is the synthesis, folding and quality control of secretory proteins. Secretory proteins usually exit the ER to enter the Golgi apparatus in coat protein complex II (COPII)-coated vesicles before transport to different subcellular destinations. However, in plants there are specialized ER-derived vesicles (ERDVs) that carry specific proteins but, unlike COPII vesicles, can exist as independent organelles or travel to the vacuole in a Golgi-independent manner. These specialized ERDVs include protein bodies and precursor-accumulating vesicles that accumulate storage proteins in the endosperm during seed development. Specialized ERDVs also include precursor protease vesicles that accumulate amino acid sequence KDEL-tailed cysteine proteases and ER bodies in Brassicales plants that accumulate myrosinases that hydrolyzes glucosinolates. These functionally specialized ERDVs act not only as storage organelles but also as platforms for signal-triggered processing, activation and deployment of specific proteins with important roles in plant growth, development and adaptive responses. Some specialized ERDVs have also been exploited to increase production of recombinant proteins and metabolites. Here we discuss our current understanding of the functional diversity, evolutionary mechanisms and biotechnological application of specialized ERDVs, which are associated with some of the highly remarkable characteristics important to plants.

NAI2 and TSA1 Drive Differentiation of Constitutive and Inducible ER Body Formation in Brassicaceae.

Brassicaceae and closely related species develop unique endoplasmic reticulum (ER)-derived structures called ER bodies, which accumulate ß-glucosidases/myrosinases that are involved in chemical defense. There are two different types of ER bodies: ER bodies constitutively present in seedlings (cER bodies) and ER bodies in rosette leaves induced by treatment with the wounding hormone jasmonate (JA) (iER bodies). Here, we show that At-α whole-genome duplication (WGD) generated the paralogous genes NAI2 and TSA1, which consequently drive differentiation of cER bodies and iER bodies in Brassicaceae plants. In Arabidopsis, NAI2 is expressed in seedlings where cER bodies are formed, whereas TSA1 is expressed in JA-treated leaves where iER bodies are formed. We found that the expression of NAI2 in seedlings and the JA inducibility of TSA1 are conserved across other Brassicaceae plants. The accumulation of NAI2 transcripts in Arabidopsis seedlings is dependent on the transcription factor NAI1, whereas the JA induction of TSA1 in rosette leaves is dependent on MYC2, MYC3 and MYC4. We discovered regions of microsynteny, including the NAI2/TSA1 genes, but the promoter regions are differentiated between TSA1 and NAI2 genes in Brassicaceae. This suggests that the divergence of function between NAI2 and TSA1 occurred immediately after WGD in ancestral Brassicaceae plants to differentiate the formation of iER and cER bodies. Our findings indicate that At-α WGD enabled diversification of defense strategies, which may have contributed to the massive diversification of Brassicaceae plants.

Dynamics of the leaf endoplasmic reticulum modulate ß-glucosidase-mediated stress-activated ABA production from its glucosyl ester.

The phytohormone abscisic acid (ABA) is produced via a multistep de novo biosynthesis pathway or via single-step hydrolysis of inactive ABA-glucose ester (ABA-GE). The hydrolysis reaction is catalyzed by ß-glucosidase (BG, or BGLU) isoforms localized to various organelles, where they become activated upon stress, but the mechanisms underlying this organelle-specific activation remain unclear. We investigated the relationship between the subcellular distribution and stress-induced activation of BGLU18 (BG1), an endoplasmic reticulum enzyme critical for abiotic stress responses, in Arabidopsis thaliana leaves. High BGLU18 levels were present in leaf petioles, primarily in endoplasmic reticulum bodies. These Brassicaceae-specific endoplasmic reticulum-derived organelles responded dynamically to abiotic stress, particularly drought-induced dehydration, by changing in number and size. Under stress, BGLU18 distribution shifted toward microsomes, which was accompanied by increasing BGLU18-mediated ABA-GE hydrolytic activity and ABA levels in leaf petioles. Under non-stress conditions, impaired endoplasmic reticulum body formation caused a microsomal shift of BGLU18 and increased its enzyme activity; however, ABA levels increased only under stress, probably because ABA-GE is supplied to the endoplasmic reticulum only under these conditions. Loss of BGLU18 delayed dehydration-induced ABA accumulation, suggesting that ABA-GE hydrolysis precedes the biosynthesis. We propose that dynamics of the endoplasmic reticulum modulate ABA homeostasis and abiotic stress responses by activating BGLU18-mediated ABA-GE hydrolysis.

PYK10 myrosinase reveals a functional coordination between endoplasmic reticulum bodies and glucosinolates in Arabidopsis thaliana.

The endoplasmic reticulum body (ER body) is an organelle derived from the ER that occurs in only three families of the order Brassicales and is suggested to be involved in plant defense. ER bodies in Arabidopsis thaliana contain large amounts of ß-glucosidases, but the physiological functions of ER bodies and these enzymes remain largely unclear. Here we show that PYK10, the most abundant ß-glucosidase in A. thaliana root ER bodies, hydrolyzes indole glucosinolates (IGs) in addition to the previously reported in vitro substrate scopolin. We found a striking co-expression between ER body-related genes (including PYK10), glucosinolate biosynthetic genes and the genes for so-called specifier proteins affecting the terminal products of myrosinase-mediated glucosinolate metabolism, indicating that these systems have been integrated into a common transcriptional network. Consistent with this, comparative metabolite profiling utilizing a number of A. thaliana relatives within Brassicaceae identified a clear phylogenetic co-occurrence between ER bodies and IGs, but not between ER bodies and scopolin. Collectively, our findings suggest a functional link between ER bodies and glucosinolate metabolism in planta. In addition, in silico three-dimensional modeling, combined with phylogenomic analysis, suggests that PYK10 represents a clade of 16 myrosinases that arose independently from the other well-documented class of six thioglucoside glucohydrolases. These findings provide deeper insights into how glucosinolates are metabolized in cruciferous plants and reveal variation of the myrosinase-glucosinolate system within individual plants.

Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein (WSCP) from Virginia pepperweed (Lepidium virginicum), a unique WSCP that preferentially binds chlorophyll b in vitro.

Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could remove Chls from the thylakoid in aqueous solution and showed an absorption spectrum identical to that of native LvWSCP. Although LvWSCP-His could bind both Chl a and Chl b, it bound almost exclusively to Chl b when reconstituted in 40 % methanol. To clarify the intracellular targeting functions of the N- and C-terminal extension peptides, we constructed transgenic Arabidopsis thaliana lines expressing the Venus protein fused with the LvWSCP N- and/or C-terminal peptides, as well as Venus fused at the C-terminus of LvWSCP. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide.

Differential contributions of two domains of NAI2 to the formation of the endoplasmic reticulum body.

The endoplasmic reticulum (ER) serves essential functions in eukaryotic cells, including protein folding, transport of secretory proteins, and lipid synthesis. The ER is a highly dynamic organelle that generates various types of compartments. Among them, the ER body is specifically present in plants in the Brassicaceae family and plays a crucial role in chemical defense against pathogens. The NAI2 protein is essential for ER body formation, and its ectopic overexpression is sufficient to induce ER body formation even in the leaves of Nicotiana benthamiana, where the ER body does not naturally exist. Despite the significance of NAI2 in ER body formation, the mechanism whereby NAI2 mediates ER body formation is not fully clear. This study aimed to investigate how two domains of Arabidopsis NAI2, the Glu-Phe-Glu (EFE) domain (ED) and the NAI2 domain (ND), contribute to ER body formation in N. benthamiana leaves. Using co-immunoprecipitation and bimolecular fluorescence complementation assays, we found that the ND is critical for homomeric interaction of NAI2 and ER body formation. Moreover, deletion of ED induced the formation of enlarged ER bodies, suggesting that ED plays a regulatory role during ER body formation. Our results indicate that the two domains of NAI2 cooperate to induce ER body formation in a balanced manner.

Texture feature extraction from microscope images enables a robust estimation of ER body phenotype in Arabidopsis.

BACKGROUND: Cellular components are controlled by genetic and physiological factors that define their shape and size. However, quantitively capturing the morphological characteristics and movement of cellular organelles from micrograph images is challenging, because the analysis deals with complexities of images that frequently lead to inaccuracy in the estimation of the features. Here we show a unique quantitative method to overcome biases and inaccuracy of biological samples from confocal micrographs. RESULTS: We generated 2D images of cell walls and spindle-shaped cellular organelles, namely ER bodies, with a maximum contrast projection of 3D confocal fluorescent microscope images. The projected images were further processed and segmented by adaptive thresholding of the fluorescent levels in the cell walls. Micrographs are composed of pixels, which have information on position and intensity. From the pixel information we calculated three types of features (spatial, intensity and Haralick) in ER bodies corresponding to segmented cells. The spatial features include basic information on shape, e.g., surface area and perimeter. The intensity features include information on mean, standard deviation and quantile of fluorescence intensities within an ER body. Haralick features describe the texture features, which can be calculated mathematically from the interrelationship between the pixel information. Together these parameters were subjected to multivariate analysis to estimate the morphological diversity. Additionally, we calculated the displacement of the ER bodies using the positional information in time-lapse images. We captured similar morphological diversity and movement within ER body phenotypes in several microscopy experiments performed in different settings and scanned under different objectives. We then described differences in morphology and movement of ER bodies between A. thaliana wild type and mutants deficient in ER body-related genes. CONCLUSIONS: The findings unexpectedly revealed multiple genetic factors that are involved in the shape and size of ER bodies in A. thaliana. This is the first report showing morphological characteristics in addition to the movement of cellular components and it quantitatively summarises plant phenotypic differences even in plants that show similar cellular components. The estimation of morphological diversity was independent of the cell staining method and the objective lens used in the microscopy. Hence, our study enables a robust estimation of plant phenotypes by recognizing small differences in complex cell organelle shapes and their movement, which is beneficial in a comprehensive analysis of the molecular mechanism for cell organelle formation that is independent of technical variations.

The Arabidopsis transcription factor NAI1 activates the NAI2 promoter by binding to the G-box motifs.

Brassicaceae plants, including Arabidopsis thaliana, develop endoplasmic reticulum (ER)-derived structures called ER bodies, which are involved in chemical defense against herbivores. NAI1 is a basic helix-loop-helix (bHLH) type transcription factor that regulates two downstream genes, NAI2 and BGLU23, that are responsible for the ER body formation and function. Here, we examined the transcription factor function of NAI1, and found that NAI1 binds to the promoter region of NAI2 and activates the NAI2 promoter. The recombinant NAI1 protein recognizes the canonical and non-canonical G-box motifs in the NAI2 promoter. Furthermore, we examined the DNA binding activity of NAI1 toward several E-box motifs in the NAI2 and BGLU23 promoters and found that NAI1 binds to a DNA fragment that includes an E-box motif from the BGLU23 promoter. Subcellular localization of NAI1 was evident in the nucleus, which is consistent with its transcription factor function. Transient expression experiments in Nicotiana benthamiana leaves showed that GFP-NAI1 protein activated the NAI2 promoter by binding to the two G-boxes of the promoter. Disruption of the G-boxes abolished the NAI1-dependent activation of the NAI2 promoter. These results indicate that NAI1 has a DNA binding activity in a motif-dependent manner and suggest that NAI1 regulates NAI2 and BGLU23 gene expressions through binding to these DNA motifs in their promoters.

Biogenesis of leaf endoplasmic reticulum body is regulated by both jasmonate-dependent and independent pathways.

Endoplasmic reticulum (ER) bodies are thought to function in plant defense against insects and pathogens. Recently, a new type of ER body referred to as "leaf ER bodies" (L-ER bodies) was identified in Arabidopsis rosette leaves. L-ER bodies accumulate two ß-glucosidases, namely PYK10 and BGLU18, which are characteristic of previously described constitutive ER bodies and inducible ER bodies, respectively. However, it is unclear how the biogenesis of L-ER bodies, which are similar to both constitutive and inducible ER bodies, is regulated. In the present study, we show that the biogenesis of L-ER bodies is regulated by both jasmonate (JA)-dependent and -independent pathways. Confocal imaging analysis revealed the presence of L-ER bodies in the JA insensitive mutant coronatine insensitive 1-1 (coi1-1), which lacks the JA receptor COI1. Quantitative reverse transcription polymerase chain reaction analysis revealed that the expression of BGLU18 mainly depends on the JA signaling pathway while that of PYK10 does not. In addition, expression of the ER body related genes NAI1, NAI2, and TSA1 was reduced in the coi1-1 mutant relative to the wild type. Taken together, these findings suggest that JA signaling is not necessary for the formation of L-ER bodies, while it is partially required for gene expression of L-ER body components.

Initiation of ER Body Formation and Indole Glucosinolate Metabolism by the Plastidial Retrograde Signaling Metabolite, MEcPP.

Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub(s) involved have remained an enigma. We have previously established that methylerythritol cyclodiphosphate (MEcPP), a precursor of plastidial isoprenoids and a stress-specific retrograde signaling metabolite, enables cellular readjustments for high-order adaptive functions. Here, we specifically show that MEcPP promotes two Brassicaceae-specific traits, namely endoplasmic reticulum (ER) body formation and induction of indole glucosinolate (IGs) metabolism selectively, via transcriptional regulation of key regulators NAI1 for ER body formation and MYB51/122 for IGs biosynthesis). The specificity of MEcPP is further confirmed by the lack of induction of wound-inducible ER body genes as well as IGs by other altered methylerythritol phosphate pathway enzymes. Genetic analyses revealed MEcPP-mediated COI1-dependent induction of these traits. Moreover, MEcPP signaling integrates the biosynthesis and hydrolysis of IGs through induction of nitrile-specifier protein1 and reduction of the suppressor, ESM1, and production of simple nitriles as the bioactive end product. The findings position the plastidial metabolite, MEcPP, as the initiation hub, transducing signals to adjust the activity of hard-wired gene circuitry to expand phytochemical diversity and alter the associated subcellular structure required for functionality of the secondary metabolites, thereby tailoring plant stress responses.

The Arabidopsis transcription factor NAI1 is required for enhancing the active histone mark but not for removing the repressive mark on PYK10, a seedling-specific gene upon embryonic-to-postgerminative developmental phase transition.

We have recently shown that the expression onset of a seedling-specific gene, PYK10, occurs in a cell-by-cell manner upon the transition from the embryonic to the postgerminative phase and during embryogenesis in seed maturation regulator mutants such as lec1, and implicated epigenetic mechanisms in the process. Here, the role of the NAI1 transcription factor required for PYK10 expression in the developmental switching of PYK10 was investigated. The cell-by-cell onset of PYK10-EGFP in lec1 embryo was still observed in the nai1 background, but at greatly reduced levels. Decreases in the level of the repressive histone mark, H3K27 trimethylation observed upon the transition to the postgeminative phase normally occurred in nai1. However, concomitant increases in the level of the active mark, H3K4 trimethylation observed in wild type was significantly compromised in nai1. These results indicate that the switching of PYK10 upon developmental phase transition involves 2 separable steps of chromatin state change.

ER bodies in plants of the Brassicales order: biogenesis and association with innate immunity.

The endoplasmic reticulum (ER) forms highly organized network structures composed of tubules and cisternae. Many plant species develop additional ER-derived structures, most of which are specific for certain groups of species. In particular, a rod-shaped structure designated as the ER body is produced by plants of the Brassicales order, which includes Arabidopsis thaliana. Genetic analyses and characterization of A. thaliana mutants possessing a disorganized ER morphology or lacking ER bodies have provided insights into the highly organized mechanisms responsible for the formation of these unique ER structures. The accumulation of proteins specific for the ER body within the ER plays an important role in the formation of ER bodies. However, a mutant that exhibits morphological defects of both the ER and ER bodies has not been identified. This suggests that plants in the Brassicales order have evolved novel mechanisms for the development of this unique organelle, which are distinct from those used to maintain generic ER structures. In A. thaliana, ER bodies are ubiquitous in seedlings and roots, but rare in rosette leaves. Wounding of rosette leaves induces de novo formation of ER bodies, suggesting that these structures are associated with resistance against pathogens and/or herbivores. ER bodies accumulate a large amount of ß-glucosidases, which can produce substances that potentially protect against invading pests. Biochemical studies have determined that the enzymatic activities of these ß-glucosidases are enhanced during cell collapse. These results suggest that ER bodies are involved in plant immunity, although there is no direct evidence of this. In this review, we provide recent perspectives of ER and ER body formation in A. thaliana, and discuss clues for the functions of ER bodies. We highlight defense strategies against biotic stress that are unique for the Brassicales order, and discuss how ER structures could contribute to these strategies.

Rice acyl-CoA-binding proteins OsACBP4 and OsACBP5 are differentially localized in the endoplasmic reticulum of transgenic Arabidopsis.

Acyl-CoA-binding proteins (ACBPs) are known to bind and transport acyl-CoA esters and phospholipids intracellularly. In our recent paper in the New Phytologist, we reported that the six acyl-CoA-binding proteins (OsACBPs) in rice (Oryza sativa) are distributed across various subcellular compartments in transgenic Arabidopsis (Arabidopsis thaliana) such as the cytosol (OsACBP1, OsACBP2 and OsACBP3), the endoplasmic reticulum (ER) including the tubules (OsACBP4 and OsACBP5) and the cisternae (OsACBP4), and the peroxisomes (OsACBP6). Localization of OsACBP4::GFP to the peripheral ER cisternae and the central cisternal ER-like structures in transgenic Arabidopsis distinguished it from OsACBP5::GFP. We further report that besides the ER, OsACBP4::GFP and OsACBP5::GFP were also targeted to the membrane of ER bodies and ER-derived spherical structures, respectively, in transgenic Arabidopsis. These findings support our previous conclusion that OsACBP4 and OsACBP5 are not redundant proteins in the ER.