The DC1 Domain Protein BINUCLEATE POLLEN is Required for POLLEN Development in Arabidopsis thaliana.

The development of the male gametophyte is a tightly regulated process that requires the precise control of cell division and gene expression. A relevant aspect to understand the events underlying pollen development regulation constitutes the identification and characterization of the genes required for this process. In this work, we showed that the DC1 domain protein BINUCLEATE POLLEN (BNP) is essential for pollen development and germination. Pollen grains carrying a defective BNP alleles failed to complete mitosis II and exhibited impaired pollen germination. By yeast two-hybrid analysis and bimolecular fluorescence complementation assays, we identified a set of BNP-interacting proteins. Among confirmed interactors, we found the NAC family transcriptional regulators Vascular Plant One-Zinc Finger 1 (VOZ1) and VOZ2. VOZ1 localization changes during pollen development, moving to the vegetative nucleus at the tricellular stage. We observed that this relocalization requires BNP; in the absence of BNP in pollen from bnp/BNP plants, VOZ1 nuclear localization is impaired. As the voz1voz2 double mutants showed the same developmental defect observed in bnp pollen grains, we propose that BNP requirement to complete microgametogenesis could be linked to its interaction with VOZ1/2 proteins. BNP could have the role of a scaffold protein, recruiting VOZ1/2 to the endosomal system into assemblies that are required for their further translocation to the nucleus, where they act as transcriptional regulators.

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.

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.

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.

Preparation, Characterization, and In Vitro Testing of Nanoclay Antimicrobial Activities and Elicitor Capacity.

Clay-based nanocomposites (nanoclays) are interesting systems to hold a wide type of active substances with a wide field of industrial applications. Bentonite-chitosan nanoclay was obtained via cationic exchange of natural bentonite (Bent) with an aqueous solution of chitosan (CS). Their physicochemical and morphological properties were discussed under the light of Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetric analysis, and scanning electron microscopy. Bent-CS characterization indicated that CS was intercalated in 10% (w/w). This polycationic polymer was oriented mostly in a monolayer arrangement, interacting by electrostatic forces between Bent sheets. The antimicrobial action of Bent-CS nanoclay was assayed onto phytopathogens, the bacterium model Pseudomonas syringe pv. tomato DC3000 ( Psy) and the necrotrophic fungus Fusarium solani f. sp. eumartii ( F. eumartii). In addition to demonstrating cell death on both microorganisms, Bent-CS exerted elicitor property on tomato plantlets. The biological actions of this natural nanomaterial might make it proper to be used in crops.

Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants.

In plants, indole-3-acetic acid (IAA) amido hydrolases (AHs) participate in auxin homeostasis by releasing free IAA from IAA-amino acid conjugates. We investigated the role of IAR3, a member of the IAA amido hydrolase family, in the response of Solanaceous plants challenged by biotrophic and hemi-biotrophic pathogens. By means of genome inspection and phylogenic analysis we firstly identified IAA-AH sequences and putative IAR3 orthologs in Nicotiana benthamiana, tomato and potato. We evaluated the involvement of IAR3 genes in defense responses by using virus-induced gene silencing. We observed that N. benthamiana and tomato plants with knocked-down expression of IAR3 genes contained lower levels of free IAA and presented altered responses to pathogen attack, including enhanced basal defenses and higher tolerance to infection in susceptible plants. We showed that IAR3 genes are consistently up-regulated in N. benthamiana and tomato upon inoculation with Phytophthora infestans and Cladosporium fulvum respectively. However, IAR3 expression decreased significantly when hypersensitive response was triggered in transgenic tomato plants coexpressing the Cf-4 resistance gene and the avirulence factor Avr4. Altogether, our results indicate that changes in IAR3 expression lead to alteration in auxin homeostasis that ultimately affects plant defense responses.

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.

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.

Phosphatidic acid formation is required for extracellular ATP-mediated nitric oxide production in suspension-cultured tomato cells.

*In animals and plants, extracellular ATP exerts its effects by regulating the second messengers Ca(2+), nitric oxide (NO) and reactive oxygen species (ROS). In animals, phospholipid-derived molecules, such as diacylglycerol, phosphatidic acid (PA) and inositol phosphates, have been associated with the extracellular ATP signaling pathway. The involvement of phospholipids in extracellular ATP signaling in plants, as it is established in animals, is unknown. *In vivo phospholipid signaling upon extracellular ATP treatment was studied in (32)P(i)-labeled suspension-cultured tomato (Solanum lycopersicum) cells. *Here, we report that, in suspension-cultured tomato cells, extracellular ATP induces the formation of the signaling lipid phosphatidic acid. Exogenous ATP at doses of 0.1 and 1 mM induce the formation of phosphatidic acid within minutes. Studies on the enzymatic sources of phosphatidic acid revealed the participation of both phospholipase D and C in concerted action with diacylglycerol kinase. *Our results suggest that extracellular ATP-mediated nitric oxide production is downstream of phospholipase C/diacylglycerol kinase 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.

Homeodomain-leucine zipper proteins interact with a plant homologue of the transcriptional co-activator multiprotein bridging factor 1.

StMBF1 (Solanum tuberosum multiprotein bridging factor 1) is a plant member of the MBF1 family of transcriptional co-activators. In an attempt to understand the role of StMBF1, we analyzed its interaction with plant transcription factors of the homeodomain-leucine zipper (Hd-Zip) family, a group of proteins with a typical leucine zipper motif adjacent to a homeodomain. StMBF1 is able to interact in vitro with the Hd-Zip protein Hahb-4 both in the presence and absence of DNA. Upon binding, StMBF1 increases the DNA binding affinity of Hahb-4, and of another plant homeodomain containing protein from the GL2/Hd-Zip IV family, HAHR-1. The biological role of interactions is discussed in this paper.

Identification of a putative Solanum tuberosum transcriptional coactivator up-regulated in potato tubers by Fusarium solani f. sp. eumartii infection and wounding.

Coadaptors or coactivators are a new class of transcription factors capable of interconnecting a regulator DNA-binding protein with a component of the basal transcription machinery allowing transcriptional activation to proceed. We report the identification of a novel Solanum tuberosum ssp. tuberosum putative transcription coactivator, named StMBF1 (Solanum tuberosum multiprotein bridging factor 1). The StMBF1 cDNA was isolated from a Fusarium solani f. sp. eumartii-infected potato tuber cDNA library, using a differential screening approach. StMBF1 is up-regulated during fungal attack as well as on wounding. A Fusarium elicitor source and ethylene precursor and salicylic acid also regulate StMBF1 expression. The precise role of StMBF1 during the plant response against environmental stresses remains to be elucidated.