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.
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 (K eq ؍ 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 K m of 41.5 M and k cat ؍ 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, Mg 2؉ , 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.
We have isolated 5 cDNA clones (din2, din6, din9, din10 and din11) corresponding to genes, the transcripts of which accumulated in leaves of Arabidopsis thaliana kept in the dark. These cDNA clones encode proteins similar to beta-glucosidase (EC 3.2.1.21, din2), asparagine synthetase (EC 6.3.5.4, din6), phosphomannose isomerase (EC 5.3.1.8, din9), seed imbibition protein (din10) and 2-oxoacid-dependent dioxygenases (din11). Accumulation of the transcripts from din6 and din10 occurred within 3 h after plants were transferred to darkness. The transcripts from din2, din9 and din11 were only detected after 24 h of dark treatment. We also observed the accumulation of the din transcripts in senescing leaves. Application of a photosynthesis inhibitor, 3-(3,4-dichlorophenyl)-1-1-dimethyl-urea, induced the expression of the din genes under illumination. Application of sucrose to detached leaves suppressed the accumulation of the din transcripts in the dark. These results indicate that expression of these genes partly depends on cellular sugar level. The sugar-modulated expression of the din genes suggests that dark-induced expression of these genes might be related to sugar starvation occurring in leaf cells in the dark, when the photosynthesis is hindered.
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.
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