Current overview of S-nitrosoglutathione (GSNO) in higher plants

S-nitrosoglutathione is a nitric oxide-derived molecule, generated by the interaction of nitric oxide (NO) with reduced glutathione (GSH) in a process called S-nitrosylation (Figure 1). The reaction appears to take place either through the formation of N2O3 or the addition of NO to a glutathionyl radical formed during this reaction (Broniowska et al., 2013). GSNO is regarded as an intracellular NO reservoir as well as a vehicle of NO throughout the cell, which enables NO biological activity to expand. GSNO is also considered to be the most abundant low-molecular-mass (LMM) S-nitrosothiol (SNO). This family includes other molecules such as S-nitrosocysteine (CySNO) and S-nitrosocysteinylglycine (GlyCySNO), which have been the subject of less study in the field of plant research. There is another group of SNOs called high-molecular mass (HMM) SNOs which are produced by NO binding to sulfhydryl (-SH) groups present in specific cysteine residues of proteins. Figure 1 shows a simple model of GSNO metabolism and its interactions with other molecules in cells where different reactions including S-nitrosylation, S-transnitrosation, and S-glutathionylation are involved (Hogg, 2002; Martínez-Ruiz and Lamas, 2007). In plants, research has focused on the importance of total SNOs in specific stress situations (Feechan et al., 2005; Chaki et al., 2011a) and on the identification of the potential protein targets of S-nitrosylation as this kind of post-translational modification can alter the function of the affected proteins (Astier et al., 2012). Initial studies in this area exogenously applied GSNO in order to identify the pool of potential protein candidates (Lindermayr et al., 2005). However, less attention has been paid to the abundance, distribution, and modulation of endogenous GSNO under natural and stress conditions. In this article, we will provide a current overview of GSNO in higher plants. [EN]Work in our laboratories is supported by ERDF-cofinanced grants from the Ministry of Science and Innovation (BIO2012-33904 and BFU2011-22779)Peer reviewe

Patterns of ROS Accumulation in the Stigmas of Angiosperms and Visions into Their Multi-Functionality in Plant Reproduction

Accumulation of reactive oxygen species (ROS) in the stigma of several plant species has been investigated. Four developmental stages (unopened flower buds, recently opened flowers, dehiscent anthers, and flowers after fertilization) were analyzed by confocal laser scanning microscopy using the ROS-specific probe DCFH2-DA. In all plants scrutinized, the presence of ROS in the stigmas was detected at higher levels during those developmental phases considered “receptive” to pollen interaction. In addition, these molecules were also present at early (unopened flower) or later (post-fertilization) stages, by following differential patterns depending on the different species. The biological significance of the presence ROS may differ between these stages, including defense functions, signaling and senescence. Pollen-stigma signaling is likely involved in the different mechanisms of self-incompatibility in these plants. The study also register a general decrease in the presence of ROS in the stigmas upon pollination, when NO is supposedly produced in an active manner by pollen grains. Finally, the distribution of ROS in primitive Angiosperms of the genus Magnolia was determined. The production of such chemical species in these plants was several orders of magnitude higher than in the remaining species evoking a massive displacement toward the defense function. This might indicate that signaling functions of ROS/NO in the stigma evolved later, as fine tune likely involved in specialized interactions like self-incompatibility.This study was supported by the following European Regional Development Fund co-financed grants: MCINN BFU2011-22779, RTC-2015-4181-2, CSIC-201540E065, CICE (Junta de Andalucía) P2010-CVI15767, P2010-AGR6274, P2011-CVI-7487, and the MINECO/CSIC agreement RECUPERA 2020.Peer reviewedPeer Reviewe

A Protocol for Minimal Single Protein Labeling with CyDye Fluors for Live Cell Internalization Assays

Individual proteins chemically labeled with fluorescent dyes can be localized and tracked in real-time experiments in order to get insights about the site and molecular mechanism of action. Here, we have adapted a protocol that was originally developed for two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) applications, to label proteins with CyDye fluors for single-molecule internalization assays in living cells. This “minimal labeling” method offers a number of advantages including specificity and known stoichiometry, simplicity, high reproducibility, and sensitivity and allows multiplexing while minimizing perturbations of the biological system. Moreover, since only a single lysine (Lys) residue per protein molecule is labeled, this method is also quantitative. To validate experimentally our protocol, we carried out the fluorescent labeling of IBB1, a major soybean protease isoinhibitor of the Bowman-Birk family that is currently being investigated as colorectal chemopreventive agent. Then, we analyzed the in vivo internalization dynamics of the labeled IBB1 protein in human colorectal adenocarcinoma HT29 cells

Análisis del proteoma del exudado estigmático

Comunicaciones a congreso

Internalization of the anti-carcinogenic IBB1, a major Bowman-Birk isoinhibitor from soybean (Glycine max), in HT29 colon cancer cells

Proceedings of the I Congress PIIISA celebrado en la Estación Experimental del Zaidín (Granada), en mayo de 2013.Protease inhibitors of the Bowman-Birk type, a major protease inhibitor family in legume seeds, which inhibit potently trypsin- and chymotrypsin-like proteases, are currently being investigated as colorectal chemopreventive agents. Although the therapeutic target/s and the action mechanism/s of Bowman-Birk inhibitors (BBI) have not yet been elucidated, the emerging evidence suggests that BBI exert their chemopreventive properties via protease inhibition; in this sense, serine proteases should be considered as primary targets in early stages of carcinogenesis. In this work, we have demonstrated that IBB1, a major protease inhibitor of the Bowman-Birk family in soybean (Glycine max), exerts anti-proliferative effect in human colorectal HT29 cancer cells at concentrations higher than 15 μM, in a dose dependent manner. By using confocal microscopy, we have demonstrated that IBB1 is taken up by HT29 colon cancer cells in a time-dependent manner, being the bulk of the internalized protease inhibitor localized in the cytoplasm where might interact with their potential therapeutic target/s.This work was supported by ERDF-co-financed grants AGL2011-26353 (Spanish Ministry of Economy and Competitiveness) and PE2010-CVI-5767 (Junta de Andalucía).Peer reviewe

Identification and localization of a caleosin in olive (Olea europaea L.) pollen during in vitro germination

In plant organs and tissues, the neutral storage lipids are confined to discrete spherical organelles called oil bodies. Oil bodies from plant seeds contain 0.6–3% proteins, including oleosins, steroleosins, and caleosins. In this study, a caleosin isoform of ∼30 kDa was identified in the olive pollen grain. The protein was mainly located at the boundaries of the oil bodies in the cytoplasm of the pollen grain and the pollen tube. In addition, caleosins were also visualized in the cytoplasm at the subapical zone, as well as in the tonoplast of vacuoles present in the pollen tube cytoplasm. The cellular behaviour of lipid bodies in the olive pollen was also monitored during in vitro germination. The number of oil bodies decreased 20-fold in the pollen grain during germination, whereas the opposite tendency occurred in the pollen tube, suggesting that oil bodies moved from one to the other. The data suggest that this pollen caleosin might have a role in the mobilization of oil bodies as well as in the reorganization of membrane compartments during pollen in vitro germination

Tomato POLLEN DEFICIENT 2 encodes a G-Type lectin receptor kinase required for viable pollen grain formation

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-Type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.This work was supported by research grants PID2019-110833RB-C31, PID2019-110833RB-C32, and PID2020-113324GB-100 funded by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033), and the Research and Innovation Programme of the European Union Horizon 2020 (BRESOV Project, ID 774244). A PhD fellowship to MGA was funded by the FPU Programme of the Spanish Ministry of Education and Culture (ref. AP2010-4528). RLe was supported by a Junta de Andalucía and FEDER research contract (DOC_01129)

A Genome-Wide Survey of Imprinted Genes in Rice Seeds Reveals Imprinting Primarily Occurs in the Endosperm

Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots

Legumes as food ingredient: characterization, processing, and applications

Editores: Jiménez-López, José Carlos (CSIC); Clemente, Alfonso (CSIC