Wheat germin-like protein: Studies on chitin/chitosan matrix for tissue engineering applications.

Advances in tissue engineering require the development of new biomaterials with adequate properties of cell attachment and growth. The properties of biomaterials can be improved by incorporation of bioactive molecules to enhance in vitro and/or in vivo functions. In this work, we study the role of a wheat germin-like protease inhibitor (GLPI), free or immobilized in biocompatible matrices to improve cell-attachment ability on different mammalian cell lines. The phylogenetic relationships and functional diversity of the GLPI were analyzed among diverse genera to get insights into sequence motif conservations. The cytocompatibility effect of free GLPI on C2C12 premyoblastic cells and B16 cells as tumoral model has been tested. GLPI promoted proliferation and metabolic activity of both cell types on in vitro models, not showing cytotoxic effects. Furthermore, GLPI was immobilized in chitin microparticles and in chitosan films; we demonstrated an accelerated cell adhesion process in both biomaterials.

Enhanced Properties of Chitosan Microparticles over Bulk Chitosan on the Modulation of the Auxin Signaling Pathway with Beneficial Impacts on Root Architecture in Plants.

Improving the root system architecture (RSA) under adverse environmental conditions by using biostimulants is emerging as a new way to boost crop productivity. Recently, we have reported the characterization of novel chitosan-based microparticles (CS-MPs) with promising biological properties as rooting agents in lettuce. In this work, we demonstrated that in contrast to bulk chitosan (CS), which exerts root growth inhibition, CS-MPs promoted root growth and development from 1 to 10 µg mL-1 without cytotoxicity effects at higher doses in Arabidopsis and lettuce seedlings. In addition, we studied the mechanistic mode of action of CS-MPs in the development of early RSA in the Arabidopsis model. CS-MPs unchained accurate and sustained spatio-temporal activation of the nuclear auxin signaling pathway. Our findings validated a promising scenario for the application of CS-MPs in the modulation of RSA to respond to changing soil environments and improve crop performance.

Nitrate reductase mediates nitric oxide-dependent gravitropic response in Arabidopsis thaliana roots.

Plant roots respond positively to gravity force and orientate it growth providing anchorage to the soil and gathering water and nutrient sources. The gravitropic response is a complex process wherein nitric oxide (NO) participates as a key signaling molecule. Here, we used genetically impaired genotypes to demonstrate the role of the nitrate reductase (NR) enzyme as a possible source of endogenous NO during gravitropic response in Arabidopsis thaliana (A. thaliana) roots. A. thaliana has two NR genes, NIA1 and NIA2. The single mutants nia1 and nia2, and the double mutant nia1/nia2 showed perturbed gravitropism. Complementation with the exogenous NO donor, S-nitroso-L-cysteine, partially rescued the wild-type phenotype in nia2 and nia1/nia2 but not in the nia1 mutant. Our findings showed that each NR gene differentially contributes to reaching the optimum level of NO during the gravitropic response, suggesting that NIA1 and NIA2 isoforms are not equivalent and have potential regulatory feedback to each other during the gravitropic response in A. thaliana roots.

Regulation of SCFTIR1/AFBs E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling.

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1-cullin-F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction.

Distribution of Endogenous NO Regulates Early Gravitropic Response and PIN2 Localization in Arabidopsis Roots.

High-resolution and automated image analysis of individual roots demonstrated that endogenous nitric oxide (NO) contribute significantly to gravitropism of Arabidopsis roots. Lowering of endogenous NO concentrations strongly reduced and even reversed gravitropism, resulting in upward bending, without affecting root growth rate. Notably, the asymmetric accumulation of NO along the upper and lower sides of roots correlated with a positive gravitropic response. Detection of NO by the specific DAF-FM DA fluorescent probe revealed that NO was higher at the lower side of horizontally-oriented roots returning to initial values 2 h after the onset of gravistimulation. We demonstrate that NO promotes plasma membrane re-localization of PIN2 in epidermal cells, which is required during the early root gravitropic response. The dynamic and asymmetric localization of both auxin and NO is critical to regulate auxin polar transport during gravitropism. Our results collectively suggest that, although auxin and NO crosstalk occurs at different levels of regulation, they converge in the regulation of PIN2 membrane trafficking in gravistimulated roots, supporting the notion that a temporally and spatially coordinated network of signal molecules could participate in the early phases of auxin polar transport during gravitropism.

Functions of S-nitrosylation in plant hormone networks.

In plants, a wide frame of physiological processes are regulated in liaison by both, nitric oxide (NO) and hormones. Such overlapping roles raise the question of how the cross-talk between NO and hormones trigger common physiological responses. In general, NO has been largely accepted as a signaling molecule that works in different processes. Among the most relevant ways NO and the NO-derived reactive species can accomplish their biological functions it is worthy to mention post-translational protein modifications. In the last years, S-nitrosylation has been the most studied NO-dependent regulatory mechanism. Briefly, S-nitrosylation is a redox-based mechanism for cysteine residue modification and is being recognized as a ubiquitous regulatory reaction comparable to phosphorylation. Therefore, it is emerging as a crucial mechanism for the transduction of NO bioactivity in plants and animals. In this mini-review, we provide an overview on S-nitrosylation of target proteins related to hormone networks in plants.

A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX.

The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3σ) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.

Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor.

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F-box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to induce auxin-regulated responses. A broad spectrum of NO-mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin-dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1-Aux/IAA interaction as evidenced by pull-down and two-hybrid assays. In addition, we provide evidence for NO-mediated modulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor. S-nitrosylation of cysteine is a redox-based post-translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1-Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S-nitrosylation enhances TIR1-Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.

Multiple p53-independent gene silencing mechanisms define the cellular response to p53 activation.

The cellular response to Nutlin-3, a small-molecule inhibitor of the p53 repressor MDM2, varies widely among human cancer-derived cell types. Whereas HCT116 colorectal carcinoma cells display sustained cell cycle arrest, BV173 leukemia cells undergo rapid apoptosis and other cell lines show an intermediate response. We found that the expression of the p53 target genes p21, 14-3-3sigma and the microRNA miR-34a correlates tightly with the cell fate choice adopted. All three genes were strongly induced in arresting cells, but silenced in cells undergoing Nutlin-3-induced apoptosis. In contrast, key apoptotic p53 target genes were equally expressed in arresting and apoptotic cells. Interestingly, we establish that miR-34a cooperates with p21 and 14-3-3sigma to override the apoptotic signals generated by p53 activation. Strikingly, p53 binding to chromatin and p53-mediated recruitment of certain coactivators to all three target loci does not vary among cell types. Instead, the cell type-specific silencing of these genes is due to enhanced p21 mRNA degradation, 14-3-3sigma promoter DNA methylation and reduced processing of the miR-34a primary transcript. Thus, p53-independent events regulating expression of protein-coding genes and microRNAs within the network can define the cellular outcome of p53 activation.

Nitric oxide promotes the wound-healing response of potato leaflets.

Nitric oxide (NO) is an essential regulatory molecule in several developmental and (patho) physiological processes. In this work, it is demonstrated that NO participates in the wound-healing response of potato leaves. The experimental approaches showed that the deposition of the cell-wall glucan callose was induced by the application of the NO donor sodium nitroprusside (SNP), and such induction was additive to the wound-induced callose production. Additionally, the expression of wound-related genes as phenylalanine ammonia-lyase (PAL) and extensin showed an accumulation of their transcript levels by SNP treatment. Moreover, the SNP-mediated increase of the PAL transcript level was additive to the induction mediated by wounding. These results indicate that increased levels of NO might potentiate the healing responses in plants leading to a rapid restoration of the damaged tissue.

Increased ratio of mitochondrial rDNA to cytoplasmic rDNA during zoosporic and germinating cyst stages of the life cycle of Phytophthora infestans (Mont.) de Bary.

A differential RNA display approach was used to study the gene expression in zoospores (Z) and germinating cysts (GC) of the late blight pathogen Phytophthora infestans. Four differentially amplified cDNAs were selected and cloned. The clone pGPiZ0.5 showed a 2.7-kb transcript highly expressed in Z. A BLAST search revealed an almost full sequence homology (98%) to the P. infestans mitochondrial large subunit rRNA. Northern blot analysis showed a twofold accumulation of the mitochondrial rRNA (mit rRNA) in Z compared with that of GC and mycelia of P. infestans. The high level of mit rRNA in Z might reflect an increased number of gene copies, an increased rDNA transcription rate, or both. Dot blot experiments indicated that the amount of mitochondrial rDNA (mit rDNA) relative to cytoplasmic rDNA is twofold higher in Z and GC than in mycelia. This relatively elevated mit rDNA could explain the high level of mit rRNA in the zoosporic phase. On the contrary, GC conserves the mit rDNA content, but the level of mit rRNA drops to 50% that of Z. The data are consistent with a very active mitochondrial protein synthesis during zoosporic phase, followed by a rapid down-regulation of mitochondrial activity during cyst formation.