Noriko Inada

Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy

Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18–0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.

Arabidopsis isochorismate synthase functional in pathogen-induced salicylate biosynthesis exhibits properties consistent with a role in diverse stress responses.

Salicylic acid (SA) is a phytohormone best known for its role in plant defense. It is synthesized in response to diverse pathogens and responsible for the large scale transcriptional induction of defense-related genes and the establishment of systemic acquired resistance. Surprisingly, given its importance in plant defense, an understanding of the underlying enzymology is lacking. In Arabidopsis thaliana, the pathogen-induced accumulation of SA requires isochorismate synthase (AtICS1). Here, we show that AtICS1 is a plastid-localized, stromal protein using chloroplast import assays and immunolocalization. AtICS1 acts as a monofunctional isochorismate synthase (ICS), catalyzing the conversion of chorismate to isochorismate (IC) in a reaction that operates near equilibrium (Keq = 0.89). It does not convert chorismate directly to SA (via an IC intermediate) as does Yersinia enterocolitica Irp9. Using an irreversible coupled spectrophotometric assay, we found that AtICS1 exhibits an apparent Km of 41.5 μm and kcat = 38.7 min-1 for chorismate. This affinity for chorismate would allow it to successfully compete with other pathogen-induced, chorismate-utilizing enzymes. Furthermore, the biochemical properties of AtICS1 indicate its activity is not regulated by light-dependent changes in stromal pH, Mg2+, or redox and that it is remarkably active at 4 °C consistent with a role for SA in cold-tolerant growth. Finally, our analyses support plastidic synthesis of stress-induced SA with the requirement for one or more additional enzymes responsible for the conversion of IC to SA, because non-enzymatic conversion of IC to SA under physiological conditions was negligible.

Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators

To elucidate host processes and components required for the sustained growth and reproduction of the obligate biotrophic fungus Golovinomyces orontii on Arabidopsis thaliana, laser microdissection was used to isolate cells at the site of infection at 5 days postinfection for downstream global Arabidopsis expression profiling. Site-specific profiling increased sensitivity dramatically, allowing us to identify specific host processes, process components, and their putative regulators hidden in previous whole-leaf global expression analyses. For example, 67 transcription factors exhibited altered expression at the powdery mildew (PM) infection site, with subsets of these playing known or inferred roles in photosynthesis, cold/dehydration responses, defense, auxin signaling, and the cell cycle. Using integrated informatics analyses, we constructed putative regulatory networks for a subset of these processes and provided strong support for host cell cycle modulation at the PM infection site. Further experimentation revealed induced host endoreduplication occurred exclusively at the infection site and led us to identify MYB3R4 as a transcriptional regulator of this process. Induced endoreduplication was abrogated in myb3r4 mutants, and G. orontii growth and reproduction were reduced. This suggests that, by increasing gene copy number, localized endoreduplication serves as a mechanism to meet the enhanced metabolic demands imposed by the fungus, which acquires all its nutrients from the plant host.

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

Abstract. The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from an imbibed seed on day 2 after sowing. Then, it matures and senesces rapidly. For analysis of the senescence pattern within individual coleoptiles, we monitored the distribution of chlorophyll (Chl) in entire coleoptiles and in cross-sections of coleoptiles by recording the autofluorescence of Chl. Degradation of Chl was apparent at the tip of the margins of opened-out coleoptiles on day 4, when the overall levels of soluble protein and Chl per coleoptile had reached maximum values. Then, senescence proceeded from the tip to the base and from the inner mesophyll cells towards the outer epidermis, excluding tissues along vascular bundles. Further analysis of cellular senescence using samples embedded in Technovit 7100 resin revealed that the senescence of each green mesophyll cell followed an identical program, which consisted of the following steps: (i) degradation of chloroplast DNA; (ii) condensation of the nucleus, decrease in the size of chloroplasts, degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast inner membranes; (iii) disorganization of the nucleus; (iv) complete loss of cellular components, distortion of the cell wall. Although the timing of each step and the rate at which each step was completed differed among cells of different locations within the coleoptile, this sequence was observed in all mesophyll cells in the coleoptile.

Novel tissue preparation method and cell-specific marker for laser microdissection of Arabidopsis mature leaf

Laser microdissection (LMD) is a powerful tool to isolate pure cell populations from heterogeneous tissues. This system has been successfully used for animal research; however, the reports of its application to plant tissues remain limited. One of the challenges of LMD for plant material is the tissue preparation. Although cryosectioning is commonly used for animal tissues, this is not a desirable method for fragile plant material with large central vacuoles. While paraffin preparation provides high histological quality and stability, the procedure is highly time consuming and may result in degradation of molecules of interest. In addition, conventional fixation and paraffin preparation methods do not preserve the structural integrity of very delicate plant tissues such as mature Arabidopsis thaliana leaves. Here, we used the rapid microwave paraffin preparation method with no fixative for preparation of Arabidopsis leaf tissue for LMD. This method resulted in Arabidopsis leaf sections with excellent preservation of leaf internal structure as evidenced by well-defined vascular bundles, phloem, and chloroplasts, and expanded and rounded epidermal cells. RNA extracted from leaf epidermal and mesophyll cells was of sufficient yield and specificity to use in downstream applications such as microarray analysis of the amplified mRNA. We employed the mesophyll cell-specific molecular marker, chloroplastic carbonic anhydrase, and developed an epidermal cell-specific marker, the very-long-chain fatty acid-condensing enzyme, CUT1, to assess specificity of harvested Arabidopsis leaf cell types by reverse transcription polymerase chain reaction. The described method is also likely to be superior for the preparation of other fragile botanical tissue for LMD and downstream applications.

Conservation and molecular dissection of ROUGH SHEATH2 and ASYMMETRIC LEAVES1 function in leaf development

Maize ROUGH SHEATH2 (RS2) and Arabidopsis ASYMMETRIC LEAVES1 (AS1) are orthologous Myb-related genes required for leaf development and act as negative regulators of class 1 KNOTTED1-like homeobox (KNOX) genes in leaf primordia. Expression of RS2 in Arabidopsis fully complements as1 leaf phenotypes and represses the expression of the KNOX gene KNAT1 in leaves. Whereas loss of AS1 function in Arabidopsis results in rounded, lobed leaves with shorter and wider petioles, overexpression of either RS2 or AS1 results in longer and narrower leaves with longer petioles than wild type. A conserved C-terminal domain (CTD) mediates homodimerization of both RS2 and AS1 and modulates leaf shape when expressed independently of the Myb domain in Arabidopsis. Homodimerization is not absolutely required for KNAT1 repression. RS2:GFP fusion protein is biologically active, localized in discrete dynamic subnuclear foci and associates with DNA during cell division.

Functional association of cell death suppressor, Arabidopsis Bax inhibitor‐1, with fatty acid 2‐hydroxylation through cytochrome b5

Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome b(5) (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation.

tie-dyed1 Regulates Carbohydrate Accumulation in Maize Leaves

Acquisition of cell identity requires communication among neighboring cells. To dissect the genetic pathways regulating cell signaling in later leaf development, a screen was performed to identify mutants with chloroplast pigmentation sectors that violate cell lineage boundaries in maize (Zea mays) leaves. We have characterized a recessive mutant, tie-dyed1 (tdy1), which develops stable, nonclonal variegated yellow and green leaf sectors. Sector formation requires high light, occurs during a limited developmental time, and is restricted to leaf blade tissue. Yellow tdy1 sectors accumulate excessive soluble sugars and starch, whereas green sectors appear unaffected. Significantly, starch accumulation precedes chlorosis in cells that will become a yellow sector. Retention of carbohydrates in tdy1 leaves is associated with a delay in reproductive maturity, decreased stature, and reduced yield. To explain the tdy1 sectoring pattern, we propose a threshold model that incorporates the light requirement and the hyperaccumulation of photoassimilates. A possible function consistent with this model is that TDY1 acts as a sugar sensor to regulate an inducible sugar export pathway as leaves develop under high light conditions.

Membrane Trafficking Pathways and their Roles in Plant–Microbe Interactions

Membrane trafficking functions in the delivery of proteins that are newly synthesized in the endoplasmic reticulum (ER) to their final destinations, such as the plasma membrane (PM) and the vacuole, and in the internalization of extracellular components or PM-associated proteins for recycling or degradative regulation. These trafficking pathways play pivotal roles in the rapid responses to environmental stimuli such as challenges by microorganisms. In this review, we provide an overview of the current knowledge of plant membrane trafficking and its roles in plant-microbe interactions. Although there is little information regarding the mechanism of pathogenic modulation of plant membrane trafficking thus far, recent research has identified many membrane trafficking factors as possible targets of microbial modulation.

Environment‐Sensitive Fluorophores with Benzothiadiazole and Benzoselenadiazole Structures as Candidate Components of a Fluorescent Polymeric Thermometer

An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537 nm) with excitation at 449 nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616 nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520 nm in n-hexane; Φ(f) =0.027, λ(em) =616 nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591 nm in n-hexane; Φ(f) =0.0046, λ(em) =672 nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.