In vivo protein kinase activity of SnRK1 fluctuates in Arabidopsis rosettes during light-dark cycles.

Sucrose-nonfermenting 1 (SNF1)-related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, and is orthologous with SNF1 in yeast and the AMP-activated protein kinase (AMPK) in animals. Previous studies of SnRK1 relied on in vitro activity assays or monitoring of putative marker gene expression. Neither approach gives unambiguous information about in vivo SnRK1 activity. We have monitored in vivo SnRK1 activity using Arabidopsis (Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with an SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle and after perturbing this cycle. As expected, in vivo SnRK1 activity rose toward the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 h into the light period. The sucrose signal metabolite, trehalose 6-phosphate (Tre6P), has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harbored an inducible trehalose-6-phosphate synthase construct. Elevated Tre6P decreased in vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose-6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, that SnRK1 activity is regulated in a context-dependent manner by Tre6P, probably interacting with further inputs including hexose phosphates and the circadian clock, and that SnRK1 signaling is modulated by factors that act downstream of SnRK1.

Impact of the SnRK1 protein kinase on sucrose homeostasis and the transcriptome during the diel cycle.

SNF1-related Kinase 1 (SnRK1) is an evolutionarily conserved protein kinase with key functions in energy management during stress responses in plants. To address a potential role of SnRK1 under favorable conditions, we performed a metabolomic and transcriptomic characterization of rosettes of 20-d-old Arabidopsis (Arabidopsis thaliana) plants of SnRK1 gain- and loss-of-function mutants during the regular diel cycle. Our results show that SnRK1 manipulation alters the sucrose and trehalose 6-phosphate (Tre6P) relationship, influencing how the sucrose content is translated into Tre6P accumulation and modulating the flux of carbon to the tricarboxylic acid cycle downstream of Tre6P signaling. On the other hand, daily cycles of Tre6P accumulation were accompanied by changes in SnRK1 signaling, leading to a maximum in the expression of SnRK1-induced genes at the end of the night, when Tre6P levels are lowest, and to a minimum at the end of the day, when Tre6P levels peak. The expression of SnRK1-induced genes was strongly reduced by transient Tre6P accumulation in an inducible Tre6P synthase (otsA) line, further suggesting the involvement of Tre6P in the diel oscillations in SnRK1 signaling. Transcriptional profiling of wild-type plants and SnRK1 mutants also uncovered defects that are suggestive of an iron sufficiency response and of a matching induction of sulfur acquisition and assimilation when SnRK1 is depleted. In conclusion, under favorable growth conditions, SnRK1 plays a role in sucrose homeostasis and transcriptome remodeling in autotrophic tissues and its activity is influenced by diel fluctuations in Tre6P levels.

Synthesis and cytotoxic evaluation of a series of 2-amino-naphthoquinones against human cancer cells.

The cytotoxicity of a series of aminonaphthoquinones resulting from the reaction of suitable aminoacids with 1,4-naphthoquinone was assayed against SF-295 (glioblastoma), MDAMB-435 (breast), HCT-8 (colon), HCT-116 (colon), HL-60 (leukemia), OVCAR-8 (ovarian), NCI-H358M (bronchoalveolar lung carcinoma) and PC3-M (prostate) cancer cells and also against PBMC (peripheral blood mononuclear cells). The results demonstrated that all the synthetic aminonaphthoquinones had relevant cytotoxic activity against all human cancer lines used in this experiment. Five of the compounds showed high cytotoxicity and selectivity against all cancer cell lines tested (IC50 = 0.49 to 3.89 µg·mL-1). The title compounds were less toxic to PBMC, since IC50 was 1.5 to eighteen times higher (IC50 = 5.51 to 17.61 µg·mL-1) than values shown by tumour cell lines. The mechanism of cell growth inhibition and structure-activity relationships remains as a target for future investigations.

Arabidopsis thaliana root elongation growth is sensitive to lunisolar tidal acceleration and may also be weakly correlated with geomagnetic variations.

BACKGROUND: Correlative evidence suggests a relationship between the lunisolar tidal acceleration and the elongation rate of arabidopsis roots grown under free-running conditions of constant low light. METHODS: Seedlings of Arabidopsis thaliana were grown in a controlled-climate chamber maintained at a constant temperature and subjected to continuous low-level illumination from fluorescent tubes, conditions that approximate to a 'free-running' state in which most of the abiotic factors that entrain root growth rates are excluded. Elongation of evenly spaced, vertical primary roots was recorded continuously over periods of up to 14 d using high temporal- and spatial-resolution video imaging and were analysed in conjunction with geophysical variables. KEY RESULTS AND CONCLUSIONS: The results confirm the lunisolar tidal/root elongation relationship. Also presented are relationships between the hourly elongation rates and the contemporaneous variations in geomagnetic activity, as evaluated from the disturbance storm time and ap indices. On the basis of time series of root elongation rates that extend over ≥4 d and recorded at different seasons of the year, a provisional conclusion is that root elongation responds to variation in the lunisolar force and also appears to adjust in accordance with variations in the geomagnetic field. Thus, both lunisolar tidal acceleration and the geomagnetic field should be considered as modulators of root growth rate, alongside other, stronger and more well-known abiotic environmental regulators, and perhaps unexplored factors such as air ions. Major changes in atmospheric pressure are not considered to be a factor contributing to oscillations of root elongation rate.

Spontaneous ultra-weak light emissions from wheat seedlings are rhythmic and synchronized with the time profile of the local gravimetric tide.

Semi-circadian rhythms of spontaneous photon emission from wheat seedlings germinated and grown in a constant environment (darkened chamber) were found to be synchronized with the rhythm of the local gravimetric (lunisolar) tidal acceleration. Time courses of the photon-count curves were also found to match the growth velocity profile of the seedlings. Pair-wise analyses of the data--growth, photon count, and tidal--by local tracking correlation always revealed significant coefficients (P > 0.7) for more than 80% of any of the time periods considered. Using fast Fourier transform, the photon-count data revealed periodic components similar to those of the gravimetric tide. Time courses of biophoton emissions would appear to be an additional, useful, and innovative tool in both chronobiological and biophysical studies.

Assessing Protein Synthesis and Degradation Rates in Arabidopsis thaliana Using Amino Acid Analysis.

Plants continually synthesize and degrade proteins, for example, to adjust protein content during development or during adaptation to new environments. In order to estimate global protein synthesis and degradation rates in plants, we developed a relatively simple and inexpensive method using a combination of 13 CO2 labeling and mass spectrometry-based analyses. Arabidopsis thaliana plants are subjected to a 24-hr 13 CO2 pulse followed by a 4-day 12 CO2 chase. Soluble alanine and serine from total protein and glucose from cell wall material are analyzed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and their 13 C enrichment (%) is estimated. The rate of protein synthesis during the 13 CO2 pulse experiment is defined as the rate of incorporation of labeled amino acids into proteins normalized by a correction factor for incomplete enrichment in free amino acid pools. The rate of protein degradation is estimated as the difference between the rate of protein synthesis and the relative growth rate calculated using the 13 C enrichment of glucose from cell wall material. Degradation rates are also estimated from the 12 CO2 pulse experiment. The following method description includes setting up and performing labeling experiments, preparation and measurement of samples, and calculation steps. In addition, an R script is provided for the calculations. 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Setting up the 13 CO2 labeling system and stable isotope labeling of Arabidopsis thaliana rosette leaves Basic Protocol 2: Extraction of soluble amino acids for GC-TOF-MS analysis Basic Protocol 3: Preparation of amino acids from total protein for GC-TOF-MS analysis Basic Protocol 4: Preparation of sugars from cell wall material for GC-TOF-MS analysis Basis Protocol 5: GC-TOF-MS analysis of 13 C-labeled samples and estimation of 13 C enrichment (%) Basis Protocol 6: Estimation of protein synthesis and degradation rates.

Lunisolar tidal synchronism with biophoton emission during intercontinental wheat-seedling germination tests.

Synchronic measurements of spontaneous ultra-weak light emission from germinating wheat seedlings both in Brazil and after transportation to Japan, and with a simultaneous series of germinations with local seedlings in the Czech Republic, are presented. A series of tests was also performed with samples returned from Japan to Brazil and results compared with those from undisturbed Brazilian seedlings. Native seedlings presented semi-circadian rhythms of emission which correlated with the gravimetric tidal acceleration at their locality, as did seeds which had been transported from Brazil to Japan, and then returned to Brazil. Here, however, there were very small disturbances within the periodicity of emissions, perhaps as a result of similar tidal profiles at locations whose longitudes are 180° apart, as in this case, different from previous results obtained in Brazil-Germany tests with other longitude shift. This feature of the Brazil and Japan locations may have minimized the requirement for the acclimatization of the transported seed to their new location.

Coincidence of biophoton emission by wheat seedlings during simultaneous, transcontinental germination tests.

Measurements of spontaneous ultra-weak light (biophoton) emission from native Brazilian and German wheat seedlings in three simultaneous series of germination tests are presented, two run in Germany and one in Brazil. Seedlings in both countries presented semi-circadian rhythms of emission that were in accordance with the local lunisolar gravimetric tidal acceleration, as did seeds which had been transported from Brazil to Germany. The simultaneity of the photon emission patterns in all tests argues for the lunisolar tide and its rhythmic variations as regulators of the natural rhythm of photon emission. However, seedlings from seed samples transported from Brazil to Germany showed, in addition, a temporary disturbance within the emission periodicity which may indicate a possible short-term acclimatization to the new location.