The Arabidopsis polyamine transporter LHR1/PUT3 modulates heat responsive gene expression by enhancing mRNA stability.

Polyamines involve in gene regulation by interacting with and modulating the functions of various anionic macromolecules such as DNA, RNA and proteins. In this study, we identified an important function of the polyamine transporter LHR1 (LOWER EXPRESSION OF HEAT RESPONSIVE GENE1) in heat-inducible gene expression in Arabidopsis thaliana. The lhr1 mutant was isolated through a forward genetic screening for altered expression of the luciferase reporter gene driven by the promoter from the heat-inducible gene AtHSP18.2. The lhr1 mutant showed reduced induction of the luciferase gene in response to heat stress and was more sensitive to high temperature than the wild type. Map-based cloning identified that the LHR1 gene encodes the polyamine transporter PUT3 (POLYAMINE UPTAKE TRANSPORTER 3) localized in the plasma membrane. The LHR1/PUT3 is required for the uptake of extracellular polyamines and plays an important role in stabilizing the mRNAs of several crucial heat stress responsive genes under high temperature. Genome-wide gene expression analysis using RNA-seq identified an array of differentially expressed genes, among which the transcript levels of some of the heat shock protein genes significantly reduced in response to prolonged heat stress in the lhr1 mutant. Our findings revealed an important heat stress response and tolerance mechanism involving polyamine influx which modulates mRNA stability of heat-inducible genes under heat stress conditions.

An investigation into the effect of potassium ions on the folding of silk fibroin studied by generalized two-dimensional NMR-NMR correlation and Raman spectroscopy.

We used generalized two-dimensional NMR-NMR correlation to examine the effect of potassium ions on the conformation transition in silk fibroin to investigate the possibility that the fairly high K+ ion content found in the distal end of silk-secreting ducts in the silkworms could have a bearing on natural formation of the silk fiber. This has enabled us to propose a detailed mechanism for the transition process. Our evidence indicates that increasing the [K+] from 0 to 3.7 mg.g(-1) in the silk fibroin, as is thought to occur as the silk fibroin moves through the secretory pathway to the spigot, produces a sequence of secondary structural changes: helix and/or random coil-->helix-like-->beta-sheet-like-->beta-sheet. The sequence is the same as that produced in silk fibroin films by decreasing the pH of fibroin from 6.8 to 4.8. In addition, we used Raman spectroscopy to study the effect of K+ ions on the Fermi doublet resonance of the tyrosyl phenolic ring at 850 and 830 cm(-1). The intensity ratio I(850)/I(830) at these wave numbers indicated that the hydrogen bonding formed by the tyrosyl phenolic-OH becomes more stable with an increase in the K+ ion concentration as above. Our investigation on the effect of K+ ions on fibroin may help provide a theoretical basis for understanding the natural silk-spinning process and the conditions required for biomimetic spinning. It may also have relevance to the aggregation of other beta-sheet proteins, including prion proteins, neurofibrillary proteins and amyloid plaques.

Generalized two-dimensional correlation analysis of NMR and Raman spectra for structural evolution characterizations of silk fibroin.

Generalized two-dimensional (2D) correlation spectroscopy was used to characterize the structural evolution of silk fibroin as the pH changed from 6.8 to 4.8, demonstrating that the conformational transitions of silk fibroin are induced step by step as the pH decreases. 2D homo- and hetero-spectral correlation spectroscopy was used to establish the relationship between information extracted from NMR and Raman spectroscopy. This novel method reveals the structural evolution using two probes with different frequency scales (10(5-9) Hz for nuclear spin motion and 10(12-14) Hz for molecular vibration motion), reflecting the different spatial scale sensitivity to the molecular conformational change. The transition order is identified as silk I state (helix dominant) --> silk I intermediate state --> silk II intermediate state --> silk II state (beta-sheet dominant), as the pH decreases. The results may rationalize the silkworm spinning process, which undergoes the conformational transition steadily from the soluble helix state to the insoluble beta-sheet state as the pH decreases from the posterior to anterior glands.