G protein signaling and metabolic pathways as evolutionarily conserved mechanisms to combat calcium deficiency.

Calcium (Ca) deficiency causes necrotic symptoms of foliar edges known as tipburn; however, the underlying cellular mechanisms have been poorly understood due to the lack of an ideal plant model and research platform. Using a phenotyping system that quantitates growth and tipburn traits in the model bryophyte Marchantia polymorpha, we evaluated metabolic compounds and the Gß-null mutant (gpb1) that modulate the occurrence and expansion of the tipburn. Transcriptomic comparisons between wild-type and gpb1 plants revealed the phenylalanine/phenylpropanoid biosynthesis pathway and reactive oxygen species (ROS) important for Ca deficiency responses. gpb1 plants reduced ROS production possibly through transcriptomic regulations of class III peroxidases and induced the expression of phenylpropanoid pathway enzymes without changing downstream lignin contents. Supplementation of intermediate metabolites and chemical inhibitors further confirmed the cell-protective mechanisms of the phenylpropanoid and ROS pathways. Marchantia polymorpha, Arabidopsis thaliana, and Lactuca sativa showed comparable transcriptomic changes where genes related to phenylpropanoid and ROS pathways were enriched in response to Ca deficiency. In conclusion, our study demonstrated unresolved signaling and metabolic pathways of Ca deficiency response. The phenotyping platform can speed up the discovery of chemical and genetic pathways, which could be widely conserved between M. polymorpha and angiosperms.

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.

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.

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.

Cryopreservation of Marchantia polymorpha spermatozoa.

The liverwort Marchantia polymorpha has become one of the model organisms, since it has less genetic redundancy, sexual and asexual modes of reproduction and a range of genomic and molecular genetic resources. Cryopreservation of fertile spermatozoa eliminates time, space and labor for growing and maintaining male plants in reproductive phase, and also provides an optional way to backup lines. Here we report a protocol to cryopreserve spermatozoa of M. polymorpha in liquid nitrogen. A cryoprotective solution containing sucrose, glycerol and egg yolk and controlled cooling and warming processes led to successful recovery of motile M. polymorpha spermatozoa after the cryogenic process. The survival rate and average motility of spermatozoa after cryopreservation were maintained at 71 and 54% of those before cryopreservation, respectively. Cryopreserved spermatozoa were capable of fertilization to form normal spores. The technique presented here confers more versatility to experiments using M. polymorpha and could be applied to preservation of plant spermatozoa in general.

Inositol Hexakis Phosphate is the Seasonal Phosphorus Reservoir in the Deciduous Woody Plant Populus alba L.

Seasonal recycling of nutrients is an important strategy for deciduous perennials. Deciduous perennials maintain and expand their nutrient pools by the autumn nutrient remobilization and the subsequent winter storage throughout their long life. Phosphorus (P), one of the most important elements in living organisms, is remobilized from senescing leaves during autumn in deciduous trees. However, it remains unknown how phosphate is stored over winter. Here we show that in poplar trees (Populus alba L.), organic phosphates are accumulated in twigs from late summer to winter, and that IP6 (myo-inositol-1,2,3,4,5,6-hexakis phosphate: phytic acid) is the primary storage form. IP6 was found in high concentrations in twigs during winter and quickly decreased in early spring. In parenchyma cells of winter twigs, P was associated with electron-dense structures, similar to globoids found in seeds of higher plants. Various other deciduous trees were also found to accumulate IP6 in twigs during winter. We conclude that IP6 is the primary storage form of P in poplar trees during winter, and that it may be a common strategy for seasonal P storage in deciduous woody plants.

Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS.

Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue.

Evolutionarily conserved regulatory mechanisms of abscisic acid signaling in land plants: characterization of ABSCISIC ACID INSENSITIVE1-like type 2C protein phosphatase in the liverwort Marchantia polymorpha.

Abscisic acid (ABA) is postulated to be a ubiquitous hormone that plays a central role in seed development and responses to environmental stresses of vascular plants. However, in liverworts (Marchantiophyta), which represent the oldest extant lineage of land plants, the role of ABA has been least emphasized; thus, very little information is available on the molecular mechanisms underlying ABA responses. In this study, we isolated and characterized MpABI1, an ortholog of ABSCISIC ACID INSENSITIVE1 (ABI1), from the liverwort Marchantia polymorpha. The MpABI1 cDNA encoded a 568-amino acid protein consisting of the carboxy-terminal protein phosphatase 2C (PP2C) domain and a novel amino-terminal regulatory domain. The MpABI1 transcript was detected in the gametophyte, and its expression level was increased by exogenous ABA treatment in the gemma, whose growth was strongly inhibited by ABA. Experiments using green fluorescent protein fusion constructs indicated that MpABI1 was mainly localized in the nucleus and that its nuclear localization was directed by the amino-terminal domain. Transient overexpression of MpABI1 in M. polymorpha and Physcomitrella patens cells resulted in suppression of ABA-induced expression of the wheat Em promoter fused to the beta -glucuronidase gene. Transgenic P. patens expressing MpABI1 and its mutant construct, MpABI1-d2, lacking the amino-terminal domain, had reduced freezing and osmotic stress tolerance, and associated with reduced accumulation of ABA-induced late embryogenesis abundant-like boiling-soluble proteins. Furthermore, ABA-induced morphological changes leading to brood cells were not prominent in these transgenic plants. These results suggest that MpABI1 is a negative regulator of ABA signaling, providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in liverworts.