Chemical and Molecular Characterization of Wound-Induced Suberization in Poplar (Populus alba × P. tremula) Stem Bark.

Upon mechanical damage, plants produce wound responses to protect internal tissues from infections and desiccation. Suberin, a heteropolymer found on the inner face of primary cell walls, is deposited in specific tissues under normal development, enhanced under abiotic stress conditions and synthesized by any tissue upon mechanical damage. Wound-healing suberization of tree bark has been investigated at the anatomical level but very little is known about the molecular mechanisms underlying this important stress response. Here, we investigated a time course of wound-induced suberization in poplar bark. Microscopic changes showed that polyphenolics accumulate 3 days post wounding, with aliphatic suberin deposition observed 5 days post wounding. A wound periderm was formed 9 days post wounding. Chemical analyses of the suberin polyester accumulated during the wound-healing response indicated that suberin monomers increased from 0.25 to 7.98 mg/g DW for days 0 to 28, respectively. Monomer proportions varied across the wound-healing process, with an overall ratio of 2:1 (monomers:glycerol) found across the first 14 days post wounding, with this ratio increasing to 7:2 by day 28. The expression of selected candidate genes of poplar suberin metabolism was investigated using qRT-PCR. Genes queried belonging to lipid polyester and phenylpropanoid metabolism appeared to have redundant functions in native and wound-induced suberization. Our data show that, anatomically, the wounding response in poplar bark is similar to that described in periderms of other species. It also provides novel insight into this process at the chemical and molecular levels, which have not been previously studied in trees.

Reconstructing the suberin pathway in poplar by chemical and transcriptomic analysis of bark tissues.

The tree bark periderm confers the first line of protection against pathogen invasion and abiotic stresses. The phellogen (cork cambium) externally produces cork (phellem) cells that are dead at maturity; while metabolically active, these tissues synthesize cell walls, as well as cell wall modifications, namely suberin and waxes. Suberin is a heteropolymer with aliphatic and aromatic domains, composed of acylglycerols, cross-linked polyphenolics and solvent-extractable waxes. Although suberin is essentially ubiquitous in vascular plants, the biochemical functions of many enzymes and the genetic regulation of its synthesis are poorly understood. We have studied suberin and wax composition in four developmental stages of hybrid poplar (Populus tremula x Populus alba) stem periderm. The amounts of extracellular ester-linked acyl lipids per unit area increased with tissue age, a trend not observed with waxes. We used RNA-Seq deep-sequencing technology to investigate the cork transcriptome at two developmental stages. The transcript analysis yielded 455 candidates for the biosynthesis and regulation of poplar suberin, including genes with proven functions in suberin metabolism, genes highlighted as candidates in other plant species and novel candidates. Among these, a gene encoding a putative lipase/acyltransferase of the GDSL-motif family emerged as a suberin polyester synthase candidate, and specific isoforms of peroxidase and laccase genes were preferentially expressed in cork, suggesting that their corresponding proteins may be involved in cross-linking aromatics to form lignin-like polyphenolics. Many transcriptional regulators with possible roles in meristem identity, cork differentiation and acyl-lipid metabolism were also identified. Our work provides the first large-scale transcriptomic dataset on the suberin-synthesizing tissue of poplar bark, contributing to our understanding of tree bark development at the molecular level. Based on these data, we have proposed a number of hypotheses that can be used in future research leading to novel biological insights into suberin biosynthesis and its physiological function.

Probing copper/tau protein interactions electrochemically.

Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid ß-peptide (Aß), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140±5mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.

Electrochemical investigations into kinase-catalyzed transformations of tau protein.

The formation of neurofibrillary tangles by hyperphosphorylated tau is a well-recognized hallmark of Alzheimer's disease. Resulting from malfunctioning protein kinases, hyperphosphorylated tau is unable to bind microtubules properly, causing it to self-associate and aggregate. The effects of tau phosphorylation on tau conformation and aggregation are still largely unexplored. The conformational analysis of tau and its hyperphosphorylated forms is usually performed by a variety of spectroscopic techniques, all of which require ample sample concentrations and/or volumes. Here we report on the use of surface based electrochemical techniques that allow for detection of conformational changes and orientation of tau protein as a function of tau phosphorylation by tyrosine and serine/threonine kinases. The electrochemical methods utilize 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative as a cosubstrate and tau immobilized on gold surface to probe the role of the following protein kinases: Sarcoma related kinase (Src), Abelson tyrosine kinase (Abl), tau-tubulin kinase (TTBK), proto-oncogene tyrosine protein kinase Fyn (Fyn), and glycogen synthase kinase 3-ß (Gsk-3ß). The single kinase and sequential kinase-catalyzed Fc-phosphorylations modulate the electrochemical signal, pointing to the dramatic changes around the Fc group in the Fc-phosphorylated tau films. The location and orientation of the Fc-group in Fc-tau film was investigated by the surface plasmon resonance based on antiferrocene antibodies. Additional surface characterization of the Fc-tau films by time-of-flight secondary ion-mass spectrometry and X-ray photoelectron spectroscopy revealed that Fc-phosphorylations influence the tau orientation and conformation on surfaces. When Fc-phosphorylations were performed in solution, the subsequently immobilized Fc-tau exhibited similar trends. This study illustrates the validity and the utility of the labeled electrochemical approach for probing the changes in protein film properties, conformation, and orientation as a function of the enzymatically catalyzed modifications.

Electrochemical investigations into Tau protein phosphorylations.

Hyperphosphorylation of Tau, a protein that stabilizes microtubules, leads to the breakdown of the microtubular structure and ultimately to the formation of neurofibrillar tangles within neurons. Here, we report monitoring of Tau phosphorylations electrochemically, using Tau protein films chemically linked to gold surfaces and 5'-γ-ferrocenyl (Fc) adenosine triphosphate (Fc-ATP) as a co-substrate. Fc-phosphorylation reactions of Tau are explored using the three protein kinases, glycogen synthase kinase (GSK-3ß), sarcoma (Src)-related kinase, and protein kinase A (PKA), which catalyze Fc-phosphorylation of different residues and regions within Tau. The kinetic parameters of the biochemical process (K(M) and V(max)) were determined.

Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.

The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 µM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.