Table_1_UV RESISTANCE LOCUS 8 From Chrysanthemum morifolium Ramat (CmUVR8) Plays Important Roles in UV-B Signal Transduction and UV-B-Induced Accumulation of Flavonoids.XLS

<p>UV Resistance Locus 8 (UVR8), an ultraviolet-B (UV-B; 280–315 nm) photoreceptor, participates in the regulation of various plant growth and developmental processes. UV-B radiation is an important factor enhancing the production of active components in medicinal plants. To-date, however, studies on UV-B photoreceptors have largely focused on Arabidopsis, and the functions of UVR8 in medicinal plants are still largely unknown. In the present study, a homolog of Arabidopsis UVR8, CmUVR8, was isolated from Chrysanthemum morifolium Ramat, and its structure and function were analyzed in detail. Protein sequence analysis showed that CmUVR8 contained nine conserved regulators of chromosome condensation 1 repeats, seven conserved bladed propellers, one C27 region, three “GWRHT” motifs and several crucial amino acid residues (such as 14 Trps and 2 Args), similar to AtUVR8. 3-D structural analysis of CmUVR8 indicated that its structure was similar to AtUVR8. Heterologous expression of CmUVR8 could rescued the deficient phenotype of uvr8-6, a mutant of UVR8 in Arabidopsis, indicating the role of CmUVR8 in the regulation of hypocotyl elongation and HY5 gene expression under UV-B irradiation. Moreover, CmUVR8 regulates UV-B-induced expression of four flavonoids biosynthesis-related genes and the UV-B-induced accumulation of flavonoids. Furthermore, the interaction between CmUVR8 and CmCOP1 were confirmed using a yeast two-hybrid assay. These results indicated that CmUVR8 plays important roles in UV-B signal transduction and the UV-B-induced accumulation of flavonoids, as a counterpart of AtUVR8.</p

Dynamics of RBC and blood plasma extravasation after laser-induced microhemorrhage.

<p>(<b>A</b>) Maximum intensity projection of a 2PEF image stack of cortical dendrites (green) and blood vessels (red), before and 1.5 hr after microhemorrhage. The spatial extent of the RBC core (blood plasma) is represented by a yellow (white) outline. (<b>B</b>) 2PEF imaging of bleeding dynamics after rupture of a single PA. A RBC-filled core (yellow outline) and diffuse plasma (white outline) expanded into the parenchyma after PA irradiation. (<b>C</b>) RBC core and (<b>D</b>) blood plasma diameter as a function of time after microhemorrhage.</p

Astrocyte activation and RBC breakdown products seven days after microhemorrhage.

<p>(<b>A</b>) Immunohistology for GFAP in coronally-sectioned tissue at lesion site and contralateral control region. (<b>B</b>) Bright-field image of coronally-sectioned tissue stained with cresyl violet (pink; neuronal cell bodies) and Prussian blue (black; RBC breakdown products).</p

Acute and chronic imaging of dendrite morphology for controls and after a deep microhemorrhage or ischemic lesion.

<p>Maximum intensity projections of 2PEF image stacks of Layer II/III cortical dendrites in YFP-H mice (<b>A</b>) in control regions, (<b>B</b>) after microhemorrhage of a single PA at 400 µm beneath the cortical surface (deep hemorrhage), and (<b>C</b>) after photothrombotic clotting of a single PA (RB clot). For the deep hemorrhage, the RBC-filled core is not visible because it was located deep in the cortex, hundreds of micrometers directly beneath the shallow dendrites imaged here.</p

Microhemorrhage size limited by clotting of vessel wall.

<p>(<b>A</b>) Median RBC core diameter as a function of time for mice receiving heparin infusion (green) and controls (red). Bold lines are running medians with 95% confidence intervals indicated by shaded areas. (<b>B</b>) RBC core diameter as a function of time for a PA that was irradiated twice (indicated by red pulses). (<b>C</b>) RBC core diameter measured at ∼90 s divided by RBC core diameter measured at ∼30 s for vessels irradiated either at 0 s (one irradiation) or at 0 s and ∼60 s (two irradiations). ***p<0.001; Mann Whitney U test.</p

Quantification of dendrite morphology after cortical microhemorrhage.

<p>(<b>A</b>) Fraction of identified dendrites that showed no signs of degeneration as a function of time for hemorrhages produced 20–100 µm beneath the cortical surface (shallow hemorrhage), 300–500 µm beneath the surface (deep hemorrhage), for photothrombotic occlusion of a PA (RB clot), and controls. (<b>B</b>) Dendrite exclusion diameter as a function of time after microhemorrhage (black). Red symbols indicate RBC core size. Error bars represent standard error of the mean (SEM). p-value compared to controls: * p<0.05, ** p<0.01, *** p<0.001, # p<0.0001, ## p<0.00001; Mann Whitney U test.</p

Compression of brain tissue near a microhemorrhage.

<p>(<b>A</b>) Maximum intensity projections of 2PEF image stacks of cortical dendrites before and 1.5 hr after a microhemorrhage. (<b>B</b>) Tissue displacement map representing the magnitude and direction of dendrite displacement after the microhemorrhage shown in panel A. Manual (red) and automated (blue) measurements are both shown. (<b>C</b>) Radially-averaged dendrite displacement as a function of distance from the center of the microhemorrhage for the example in panels A and B. (<b>D</b>) Average dendrite displacement (black circles) and fit to Equation (10) (green line) as a function of normalized distance from the center of the microhemorrhage.</p

Acute and chronic imaging of microglia/macrophage response after microhemorrhage.

<p>(<b>A</b>) Maximum intensity projection of 2PEF image stacks of microglia/macrophages (green) and blood vessels (red) before and after microhemorrhage (left). Chronic dynamics of microglia/macrophage behavior after microhemorrhage (right). Inset at right shows reactive processes invading the RBC-filled core (indicated by yellow outline) at 1.5 hr after the lesion. (<b>B</b>) Microglia/macrophage density relative to baseline at different distances from the microhemorrhage as a function of time for homozygous and heterozygous CX₃CR1-GFP mice (p-value compared to control: * <0.05, †† <0.01; analysis of covariance). (<b>C</b>) Maximal distance from the center of the microhemorrhage to the furthest responsive microglia/macrophage as a function of time after the lesion. Red symbol indicates distance to furthest responsive cell after laser ablation in the cortical parenchyma with an energy similar to that used to induce a microhemorrhage. (<b>D</b>) Fraction of microglia/macrophages with processes directed toward the lesion after a microhemorrhage (p-value compared to control for homozygous (heterozygous) mice: * (†) p<0.05, ** (††) p<0.01, *** (†††) p<0.001, # (%) p<0.0001, ## (%%) p<0.00001; Mann Whitney U test). (<b>E</b>) Diameter of region where microglia/macrophages are excluded after microhemorrhage as a function of time. Average RBC core diameter for the heterozygous (homozygous) animals is indicated in green (red) at 0.5 and 1.5 hr post hemorrhage. Error bars represent the standard error of the mean (SEM).</p

Acute and chronic imaging of dendrite morphology after shallow microhemorrhage.

<p>Maximum intensity projections of 2PEF image stacks of Layer II/III cortical dendrites from YPF-H mice at different times after microhemorrhage of a single PA 30 µm beneath the cortical surface (shallow hemorrhage).</p

Microhemorrhages do not crush nearby capillaries, but blood flow speed is reduced.

<p>(<b>A</b>) Maximum intensity projections of 2PEF image stacks of blood vessels in the vicinity of the RBC core (yellow outline) before and 1.5 hr after a microhemorrhage. (<b>B</b>) Classification of capillary segments within 125 µm from the target PA, identified before the lesion, as flowing, stalled, or missing at 1.5 hr after the lesion. Error bars represent binomial 95% confidence intervals. (<b>C</b>) 2PEF images and space-time linescans of a capillary located ∼70 µm from the target PA before and 1.5 hr after a microhemorrhage. (<b>D</b>) Boxplot of capillary blood flow speed 1.5 hr after a microhemorrhage, expressed as a fraction of the baseline speed for capillaries within 125 µm from the target PA. In control measurements, no hemorrhage was produced. ***p<0.001; Mann Whitney U test.</p