Integration of cell differentiation and initiation of monoterpenoid indole alkaloid metabolism in seed germination of Catharanthus roseus.

In Catharanthus roseus, monoterpenoid indole alkaloids (MIAs) are produced through the cooperation of four cell types, with final products accumulating in specialized cells known as idioblasts and laticifers. To explore the relationship between cellular differentiation and cell type-specific MIA metabolism, we analyzed the expression of MIA biosynthesis in germinating seeds. Embryos from immature and mature seeds were observed via stereomicroscopy, fluorescence microscopy, and electron microscopy. Time-series MIA and iridoid quantification, along with transcriptome analysis, were conducted to determine the initiation of MIA biosynthesis. In addition, the localization of MIAs was examined using alkaloid staining and imaging mass spectrometry (IMS). Laticifers were present in embryos before seed maturation. MIA biosynthesis commenced 12 h after germination. MIAs accumulated in laticifers of embryos following seed germination, and MIA metabolism is induced after germination in a tissue-specific manner. These findings suggest that cellular morphological differentiation precedes metabolic differentiation. Considering the well-known toxicity and defense role of MIAs in matured plants, MIAs may be an important defense strategy already in the delicate developmental phase of seed germination, and biosynthesis and accumulation of MIAs may require the tissue and cellular differentiation.

Phosphate environment and phosphate uptake studies: past and future.

The present review explains briefly the importance of phosphorus in the biological activities and states that the most phosphorus of living organisms is absorbed by plants from the soil. Next, previous studies on the mechanisms of phosphate uptake by plants are reviewed as H+-dependent or Na+-dependent co-transport systems and the phosphate environment in which plants grow is discussed. The evolution of transporter genes and their regulation mechanisms of expression is discussed in relation to the phosphorus environment.

Temperature-Regulated Flowering Locus T-Like Gene Coordinates the Spike Initiation in Phalaenopsis Orchid.

Phalaenopsis aphrodite can be induced to initiate spike growth and flowering by exposure to low ambient temperatures. However, the factors and mechanisms responsible for spike initiation in P. aphrodite remain largely unknown. In this study, we show that a repressor Flowing Locus T-like (FTL) gene, FTL, can act as a negative regulator of spike initiation in P. aphrodite. The mRNA transcripts of PaFTL are consistently high during high ambient temperature, thereby preventing premature spike initiation. However, during low ambient temperature, PaFTL expression falls while FT expression increases, allowing for spike initiation. Knock-down of PaFTL expression through virus-inducing gene silencing promoted spike initiation at 30/28°C. Moreover, PaFTL interacts with FLOWERING LOCUS D in a similar manner to FT to regulate downstream flowering initiation genes. Transgenic P. aphrodite plants exhibiting high expression of PaFTL do not undergo spike initiation, even when exposed to low ambient temperatures. These findings shed light on the flowering mechanisms in Phalaenopsis and provide new insights into how perennial plants govern spike initiation in response to temperature cues.

Uncovering molecular mechanisms involved in microbial volatile compounds-induced stomatal closure in Arabidopsis thaliana.

Microbial volatile compounds (mVCs) may cause stomatal closure to limit pathogen invasion as part of plant innate immune response. However, the mechanisms of mVC-induced stomatal closure remain unclear. In this study, we co-cultured Enterobacter aerogenes with Arabidopsis (Arabidopsis thaliana) seedlings without direct contact to initiate stomatal closure. Experiments using the reactive oxygen species (ROS)-sensitive fluorescent dye, H2DCF-DA, showed that mVCs from E. aerogenes enhanced ROS production in guard cells of wild-type plants. The involvement of ROS in stomatal closure was then demonstrated in an ROS production mutant (rbohD). In addition, we identified two stages of signal transduction during E. aerogenes VC-induced stomatal closure by comparing the response of wild-type Arabidopsis with a panel of mutants. In the early stage (3 h exposure), E. aerogenes VCs induced stomatal closure in wild-type and receptor-like kinase THESEUS1 mutant (the1-1) but not in rbohD, plant hormone-related mutants (nced3, erf4, jar1-1), or MAPK kinase mutants (mkk1 and mkk3). However, in the late stage (24 h exposure), E. aerogenes VCs induced stomatal closure in wild-type and rbohD but not in nced3, erf4, jar1-1, the1-1, mkk1 or mkk3. Taken together, our results suggest that E. aerogenes mVC-induced plant immune responses modulate stomatal closure in Arabidopsis by a multi-phase mechanism.

Genetic Interaction between Arabidopsis SUR2/CYP83B1 and GNOM Indicates the Importance of Stabilizing Local Auxin Accumulation in Lateral Root Initiation.

Lateral root (LR) formation is an important developmental event for the establishment of the root system in most vascular plants. In Arabidopsis thaliana, the fewer roots (fwr) mutation in the GNOM gene, encoding a guanine nucleotide exchange factor of ADP ribosylation factor that regulates vesicle trafficking, severely inhibits LR formation. Local accumulation of auxin response for LR initiation is severely affected in fwr. To better understand how local accumulation of auxin response for LR initiation is regulated, we identified a mutation, fewer roots suppressor1 (fsp1), that partially restores LR formation in fwr. The gene responsible for fsp1 was identified as SUPERROOT2 (SUR2), encoding CYP83B1 that positions at the metabolic branch point in the biosynthesis of auxin/indole-3-acetic acid (IAA) and indole glucosinolate. The fsp1 mutation increases both endogenous IAA levels and the number of the sites where auxin response locally accumulates prior to LR formation in fwr. SUR2 is expressed in the pericycle of the differentiation zone and in the apical meristem in roots. Time-lapse imaging of the auxin response revealed that local accumulation of auxin response is more stable in fsp1. These results suggest that SUR2/CYP83B1 affects LR founder cell formation at the xylem pole pericycle cells where auxin accumulates. Analysis of the genetic interaction between SUR2 and GNOM indicates the importance of stabilization of local auxin accumulation sites for LR initiation.

The involvement of AtMKK1 and AtMKK3 in plant-deleterious microbial volatile compounds-induced defense responses.

KEY MESSAGE: Plant-deleterious microbial volatiles activate the transactivation of hypoxia, MAMPs and wound responsive genes in Arabidopsis thaliana. AtMKK1 and AtMKK3 are involved in the plant-deleterious microbial volatiles-induced defense responses. Microbial volatile compounds (mVCs) are a collection of volatile metabolites from microorganisms with biological effects on all living organisms. mVCs function as gaseous modulators of plant growth and plant health. In this study, the defense events induced by plant-deleterious mVCs were investigated. Enterobacter aerogenes VCs lead to growth inhibition and immune responses in Arabidopsis thaliana. E. aerogenes VCs negatively regulate auxin response and transport gene expression in the root tip, as evidenced by decreased expression of DR5::GFP, PIN3::PIN3-GFP and PIN4::PIN4-GFP. Data from transcriptional analysis suggests that E. aerogenes VCs trigger hypoxia response, innate immune responses and metabolic processes. In addition, the transcript levels of the genes involved in the synthetic pathways of antimicrobial metabolites camalexin and coumarin are increased after the E. aerogenes VCs exposure. Moreover, we demonstrate that MKK1 serves as a regulator of camalexin biosynthesis gene expression in response to E. aerogenes VCs, while MKK3 is the regulator of coumarin biosynthesis gene expression. Additionally, MKK1 and MKK3 mediate the E. aerogenes VCs-induced callose deposition. Collectively, these studies provide molecular insights into immune responses by plant-deleterious mVCs.

Elastic shell theory for plant cell wall stiffness reveals contributions of cell wall elasticity and turgor pressure in AFM measurement.

The stiffness of a plant cell in response to an applied force is determined not only by the elasticity of the cell wall but also by turgor pressure and cell geometry, which affect the tension of the cell wall. Although stiffness has been investigated using atomic force microscopy (AFM) and Young's modulus of the cell wall has occasionally been estimated using the contact-stress theory (Hertz theory), the existence of tension has made the study of stiffness more complex. Elastic shell theory has been proposed as an alternative method; however, the estimation of elasticity remains ambiguous. Here, we used finite element method simulations to verify the formula of the elastic shell theory for onion (Allium cepa) cells. We applied the formula and simulations to successfully quantify the turgor pressure and elasticity of a cell in the plane direction using the cell curvature and apparent stiffness measured by AFM. We conclude that tension resulting from turgor pressure regulates cell stiffness, which can be modified by a slight adjustment of turgor pressure in the order of 0.1 MPa. This theoretical analysis reveals a path for understanding forces inherent in plant cells.

Imaging MS Analysis in Catharanthus roseus.

To understand how the plant regulates metabolism, it is important to determine where metabolites localize in the tissues and cells. Single-cell level omics approaches in plants have shown remarkable development over the last several years, and this data has been instrumental in gene discovery efforts for enzymes and transporters involved in metabolism. For metabolomics, Imaging Mass Spectrometry (IMS) is a powerful tool to map the spatial distribution of molecules in the tissue. Here, we describe the methods which we used to reveal where secondary metabolites, primarily alkaloids, localize in Catharanthus roseus stem and leaf tissues.

Visualization of phosphorus re-translocation and phosphate transporter expression profiles in a shortened annual cycle system of poplar.

Phosphorus (P) is an essential macronutrient for plant growth. In deciduous trees, P is remobilized from senescing leaves and stored in perennial tissues during winter for further growth. Annual internal recycling and accumulation of P are considered an important strategy to support the vigorous growth of trees. However, the pathways of seasonal re-translocation of P and the molecular mechanisms of this transport have not been clarified. Here we show the seasonal P re-translocation route visualized using real-time radioisotope imaging and the macro- and micro-autoradiography. We analysed the seasonal re-translocation P in poplar (Populus alba. L) cultivated under 'a shortened annual cycle system', which mimicked seasonal phenology in a laboratory. From growing to senescing season, sink tissues of 32 P and/or 33 P shifted from young leaves and the apex to the lower stem and roots. The radioisotope P re-translocated from a leaf was stored in phloem and xylem parenchyma cells and redistributed to new shoots after dormancy. Seasonal expression profile of phosphate transporters (PHT1, PHT5 and PHO1 family) was obtained in the same system. Our results reveal the seasonal P re-translocation routes at the organ and tissue levels and provide a foothold for elucidating its molecular mechanisms.

Differential regulation of fluorescent alkaloid metabolism between idioblast and lacticifer cells during leaf development in Catharanthus roseus seedlings.

Bioactive specialized (secondary) metabolites are indispensable for plant development or adjustment to their surrounding environment. In many plants, these specialized metabolites are accumulated in specifically differentiated cells. Catharanthus roseus is a well-known medicinal plant known for producing many kinds of monoterpenoid indole alkaloids (MIAs). C. roseus has two types of specifically differentiated cells accumulating MIAs, so-called idioblast cells and laticifer cells. In this study, we compared each of the cells as they changed during seedling growth, and found that the fluorescent metabolites accumulated in these cells were differentially regulated. Analysis of fluorescent compounds revealed that the fluorescence observed in these cells was emitted from the compound serpentine. Further, we found that the serpentine content of leaves increased as leaves grew. Our findings suggest that idioblast cells and laticifer cells have different biological roles in MIA biosynthesis and its regulation.

Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus.

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the ß-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.

Calcium dynamics during trap closure visualized in transgenic Venus flytrap.

The leaves of the carnivorous plant Venus flytrap, Dionaea muscipula (Dionaea) close rapidly to capture insect prey. The closure response usually requires two successive mechanical stimuli to sensory hairs on the leaf blade within approximately 30 s (refs. 1-4). An unknown biological system in Dionaea is thought to memorize the first stimulus and transduce the signal from the sensory hair to the leaf blade2. Here, we link signal memory to calcium dynamics using transgenic Dionaea expressing a Ca2+ sensor. Stimulation of a sensory hair caused an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) starting in the sensory hair and spreading to the leaf blade. A second stimulus increased [Ca2+]cyt to an even higher level, meeting a threshold that is correlated to the leaf blade closure. Because [Ca2+]cyt gradually decreased after the first stimulus, the [Ca2+]cyt increase induced by the second stimulus was insufficient to meet the putative threshold for movement after about 30 s. The Ca2+ wave triggered by mechanical stimulation moved an order of magnitude faster than that induced by wounding in petioles of Arabidopsis thaliana5 and Dionaea. The capacity for rapid movement has evolved repeatedly in flowering plants. This study opens a path to investigate the role of Ca2+ in plant movement mechanisms and their evolution.

GEMMA CUP-ASSOCIATED MYB1, an Ortholog of Axillary Meristem Regulators, Is Essential in Vegetative Reproduction in Marchantia polymorpha.

A variety of plants in diverse taxa can reproduce asexually via vegetative propagation, in which clonal propagules with a new meristem(s) are generated directly from vegetative organs. A basal land plant, Marchantia polymorpha, develops clonal propagules, gemmae, on the gametophyte thallus from the basal epidermis of a specialized receptacle, the gemma cup. Here we report an R2R3-MYB transcription factor, designated GEMMA CUP-ASSOCIATED MYB1 (GCAM1), which is an essential regulator of gemma cup development in M. polymorpha. Targeted disruption of GCAM1 conferred a complete loss of gemma cup formation and gemma generation. Ectopic overexpression of GCAM1 resulted in formation of cell clumps, suggesting a function of GCAM1 in suppression of cell differentiation. Although gemma cups are a characteristic gametophyte organ for vegetative reproduction in a taxonomically restricted group of liverwort species, phylogenetic and interspecific complementation analyses support the orthologous relationship of GCAM1 to regulatory factors of axillary meristem formation, e.g., Arabidopsis REGULATOR OF AXILLARY MERISTEMS and tomato Blind, in angiosperm sporophytes. The present findings in M. polymorpha suggest an ancient acquisition of a transcriptional regulator for production of asexual propagules in the gametophyte and the use of the regulatory factor for diverse developmental programs, including axillary meristem formation, during land plant evolution.

The RopGEF KARAPPO Is Essential for the Initiation of Vegetative Reproduction in Marchantia polymorpha.

Many plants can reproduce vegetatively, producing clonal progeny from vegetative cells; however, little is known about the molecular mechanisms underlying this process. Liverwort (Marchantia polymorpha), a basal land plant, propagates asexually via gemmae, which are clonal plantlets formed in gemma cups on the dorsal side of the vegetative thallus [1]. The initial stage of gemma development involves elongation and asymmetric divisions of a specific type of epidermal cell, called a gemma initial, which forms on the floor of the gemma cup [2, 3]. To investigate the regulatory mechanism underlying gemma development, we focused on two allelic mutants in which no gemma initial formed; these mutants were named karappo, meaning "empty." We used whole-genome sequencing of both mutants and molecular genetic analysis to identify the causal gene, KARAPPO (KAR), which encodes a ROP guanine nucleotide exchange factor (RopGEF) carrying a plant-specific ROP nucleotide exchanger (PRONE) catalytic domain. In vitro GEF assays showed that the full-length KAR protein and the PRONE domain have significant GEF activity toward MpROP, the only ROP GTPase in M. polymorpha. Moreover, genetic complementation experiments showed a significant role for the N- and C-terminal variable regions in gemma development. Our investigation demonstrates an essential role for KAR/RopGEF in the initiation of plantlet development from a differentiated cell, which may involve cell-polarity formation and subsequent asymmetric cell division via activation of ROP signaling, implying a similar developmental mechanism in vegetative reproduction of various land plants.

The complexity of intercellular localisation of alkaloids revealed by single-cell metabolomics.

Catharanthus roseus is a medicinal plant well known for producing bioactive compounds such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). Although the leaves of this plant are the main source of these antitumour drugs, much remains unknown on how TIAs are biosynthesised from a central precursor, strictosidine, to various TIAs in planta. Here, we have succeeded in showing, for the first time in leaf tissue of C. roseus, cell-specific TIAs localisation and accumulation with 10 µm spatial resolution Imaging mass spectrometry (Imaging MS) and live single-cell mass spectrometry (single-cell MS). These metabolomic studies revealed that most TIA precursors (iridoids) are localised in the epidermal cells, but major TIAs including serpentine and vindoline are localised instead in idioblast cells. Interestingly, the central TIA intermediate strictosidine also accumulates in both epidermal and idioblast cells of C. roseus. Moreover, we also found that vindoline accumulation increases in laticifer cells as the leaf expands. These discoveries highlight the complexity of intercellular localisation in plant specialised metabolism.

Lateral root initiation requires the sequential induction of transcription factors LBD16 and PUCHI in Arabidopsis thaliana.

Lateral root (LR) formation in Arabidopsis thaliana is initiated by asymmetric division of founder cells, followed by coordinated cell proliferation and differentiation for patterning new primordia. The sequential developmental processes of LR formation are triggered by a localized auxin response. LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), an auxin-inducible transcription factor, is one of the key regulators linking auxin response in LR founder cells to LR initiation. We identified key genes for LR formation that are activated by LBD16 in an auxin-dependent manner. LBD16 targets identified include the transcription factor gene PUCHI, which is required for LR primordium patterning. We demonstrate that LBD16 activity is required for the auxin-inducible expression of PUCHI. We show that PUCHI expression is initiated after the first round of asymmetric cell division of LR founder cells and that premature induction of PUCHI during the preinitiation phase disrupts LR primordium formation. Our results indicate that LR initiation requires the sequential induction of transcription factors LBD16 and PUCHI.

Lateral Inhibition by a Peptide Hormone-Receptor Cascade during Arabidopsis Lateral Root Founder Cell Formation.

In plants, the position of lateral roots (LRs) depends on initiation sites induced by auxin. The domain of high auxin response responsible for LR initiation stretches over several cells, but only a pair of pericycle cells (LR founder cells) will develop into LRs. In this work, we identified a signaling cascade controlling LR formation through lateral inhibition. It comprises a peptide hormone TARGET OF LBD SIXTEEN 2 (TOLS2), its receptor RLK7, and a downstream transcription factor PUCHI. TOLS2 is expressed at the LR founder cells and inhibits LR initiation. Time-lapse imaging of auxin-responsive DR5:LUCIFERASE reporter expression revealed that occasionally two pairs of LR founder cells are specified in close proximity even in wild-type and that one of them exists only transiently and disappears in an RLK7-dependent manner. We propose that the selection of LR founder cells by the peptide hormone-receptor cascade ensures proper LR spacing.

Organelle DNA degradation contributes to the efficient use of phosphate in seed plants.

Mitochondria and chloroplasts (plastids) both harbour extranuclear DNA that originates from the ancestral endosymbiotic bacteria. These organelle DNAs (orgDNAs) encode limited genetic information but are highly abundant, with multiple copies in vegetative tissues, such as mature leaves. Abundant orgDNA constitutes a substantial pool of organic phosphate along with RNA in chloroplasts, which could potentially contribute to phosphate recycling when it is degraded and relocated. However, whether orgDNA is degraded nucleolytically in leaves remains unclear. In this study, we revealed the prevailing mechanism in which organelle exonuclease DPD1 degrades abundant orgDNA during leaf senescence. The DPD1 degradation system is conserved in seed plants and, more remarkably, we found that it was correlated with the efficient use of phosphate when plants were exposed to nutrient-deficient conditions. The loss of DPD1 compromised both the relocation of phosphorus to upper tissues and the response to phosphate starvation, resulting in reduced plant fitness. Our findings highlighted that DNA is also an internal phosphate-rich reservoir retained in organelles since their endosymbiotic origin.

Molecular Components of Arabidopsis Intact Vacuoles Clarified with Metabolomic and Proteomic Analyses.

We analyzed the metabolites and proteins contained in pure intact vacuoles isolated from Arabidopsis suspension-cultured cells using capillary electrophoresis-mass spectrometry (CE-MS), Fourier transform-ion cyclotron resonance (FT-ICR)-MS and liquid chromatography (LC)-MS. We identified 21 amino acids and five organic acids as major primary metabolites in the vacuoles with CE-MS. Further, we identified small amounts of 27 substances including well-known vacuolar molecules, but also some unexpected substances (e.g. organic phosphate compounds). Non-target analysis of the vacuolar sample with FT-ICR-MS suggested that there are 1,106 m/z peaks that could predict the 5,090 molecular formulae, and we have annotated 34 compounds in these peaks using the KNapSAck database. By conducting proteomic analysis of vacuolar sap, we found 186 proteins in the same vacuole samples. Since the vacuole is known as a major degradative compartment, many of these were hydrolases, but we also found various oxidoreductases and transferases. The relationships between the proteins and metabolites in the vacuole are discussed.