Striated muscle is a highly organized structure composed of well-defined anatomical domains with integrated but distinct assignments. So far, the lack of a direct correlation between tissue ...architecture and gene expression has limited our understanding of how each unit responds to physio-pathologic contexts. Here, we show how the combined use of spatially resolved transcriptomics and immunofluorescence can bridge this gap by enabling the unbiased identification of such domains and the characterization of their response to external perturbations. Using a spatiotemporal analysis, we follow changes in the transcriptome of specific domains in muscle in a model of denervation. Furthermore, our approach enables us to identify the spatial distribution and nerve dependence of atrophic signaling pathway and polyamine metabolism to glycolytic fibers. Indeed, we demonstrate that perturbations of polyamine pathway can affect muscle function. Our dataset serves as a resource for future studies of the mechanisms underlying skeletal muscle homeostasis and innervation.
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•Characterization of functional cluster in adult muscle using spatial transcriptomics•Identification of differentially activated pathway during denervation•Polyamine pathway is spatially restricted and innervation responsive
Using spatially resolved transcriptomics, D’Ercole et al. have analyzed the different morphofunctional domains in muscle and their response to reversible nerve damage, highlighting the polyamine pathway as a potential contributor to the atrophic response.
In
Solanum Melongena L. Cv. Pusa Purple Long, Significant Differences Were Observed For Embryogenic Potential Within A Single Explant (Hypocotyl System). Terminal Hypocotyl Segments (Apical and ...Basal) Yielded A Higher Number Of Somatic Embryos Than The Medial (Sub-Apical and Sub-Basal) Segments. High Levels Of Conjugated Spermidine Along With High Levels Of Total Polyamines (Primarily Free Fraction) Could Be Correlated With The Formation Of Somatic Embryos In Terminal Segments. Temporal Changes In Endogenous Levels Of Free, Conjugated and Bound Putrescine, Spermidine and Spermine Were Analyzed At Critical Stages Of Somatic Embryogenesis From Four Different Hypocotyl Segments. Within 3 Days Of Culture, There Was A Sharp Decline In Free (and Total) Putrescine and An Increase In Conjugated Spermidine Levels. All Hypocotyl Segments Attained Similar Levels Of Free Spermidine, Irrespective Of Their Subsequent Embryogenic Response. As The Tissues Become Committed For Embryogenesis, Free, Conjugated and Total Putrescine and Spermidine Reach Uniformly Minimal Levels For All Segments. Just Before The Onset Of Embryogenesis, There Was A Dramatic Increase In All Fractions Of Putrescine, and In Free and Conjugated (Along With Total) Spermidine Levels. Intermediate Levels Of Conjugated and Total Putrescine and Spermidine Were Reached In Tissues With Good Embryogenic Potential: tissues with poor embryogenic potential attained lower or higher levels. After the embryos were formed, their levels fell sharply, and continued to decline. Free putrescine, however, reached another peak when embryos had fully matured. The changes in bound putrescine and spermidine, and in spermine (all forms) could not be correlated with somatic embryogenesis.
Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from ...root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave Km values for putrescine and S-adenosyl-L-methionine of 0.31 mM and 0.10 mM, respectively, and Ki values for S-adenosyl-L-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a Ki of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.
Transformed root cultures of Datura stramonium, competent in tropane-alkaloid biosynthesis, have been treated with exogenous plant growth regulators. It was found that combinations of ...α-naphthalene-acetic acid, kinetin (N6-furfurylaminopurine) and 2,4-dichlorophenoxyacetic acid induced de-differentiation, causing both the rooty phenotype and the hyoscyamine-biosynthetic capacity to be lost. Alkaloid biosynthesis disappeared rapidly and prior to the loss of morphological integrity. It was observed that the enzymes ornithine decarboxylase (EC 4.1.1.17), arginine decarboxylase (EC 4.1.1.19) and N-methylputrescine oxidase did not show the increase in level normally associated with subculturing the roots. The level of putrescine N-methyltransferase (EC 2.1.1.53) activity, the first enzyme fully committed to hyoscyamine biosynthesis, rapidly declined, about 80% being lost from the roots within 12 h. This activity, although showing some temporary restoration, declined further after a few days, and was totally absent from fully dispersed cultures. N-Methylputrescine oxidase persisted at a low level. Following sub-culture of established de-differentiated lines to plant-growth-regulator-free medium, limited root regeneration occurred. The roots formed showed renewed competence in alkaloid biosynthesis and putrescine N-methyltransferase and N-methylputrescine oxidase activities were restored to their normal levels. The relationship between the morphological state and alkaloid-biosynthetic capacity of the cultures is discussed in relation to the overall control of alkaloid biosynthesis.
Poplar shoots raised
in vitro rooted by 100 °lo in the presence of an auxin while in its absence they did not root. Putrescine, however, and the inhibitor of spermidine synthase, cyclohexylamine ...(CHA), which favours putrescine accumulation, were able to promote up to 40 % rooting in the absence of auxin. The inhibitory effect of aminoguanidine (AG), which inhibits diamine oxidase, indicated that the catabolic pathway of putrescine was involved in the rooting process. The inductive phase of rooting in poplar shoots was characterized by an increase of peroxidase activity followed by a rapid decrease. Putrescine and CHA favoured this variation. AG and spermidine had adversary effects.
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