The Common Bean Small Heat Shock Protein Nodulin 22 from Phaseolus vulgaris L. Assembles into Functional High-Molecular-Weight Oligomers.

Small heat shock proteins (sHsps) are present in all domains of life. These proteins are responsible for binding unfolded proteins to prevent their aggregation. sHsps form dynamic oligomers of different sizes and constitute transient reservoirs for folding competent proteins that are subsequently refolded by ATP-dependent chaperone systems. In plants, the sHsp family is rather diverse and has been associated with the ability of plants to survive diverse environmental stresses. Nodulin 22 (PvNod22) is an sHsp of the common bean (Phaseolus vulgaris L.) located in the endoplasmic reticulum. This protein is expressed in response to stress (heat or oxidative) or in plant roots during mycorrhizal and rhizobial symbiosis. In this work, we study its oligomeric state using a combination of in silico and experimental approaches. We found that recombinant PvNod22 was able to protect a target protein from heat unfolding in vitro. We also demonstrated that PvNod22 assembles into high-molecular-weight oligomers with diameters of ~15 nm under stress-free conditions. These oligomers can cluster together to form high-weight polydisperse agglomerates with temperature-dependent interactions; in contrast, the oligomers are stable regarding temperature.

Mayahuelin, a Type I Ribosome Inactivating Protein: Characterization, Evolution, and Utilization in Phylogenetic Analyses of Agave.

Agaves resist extreme heat and drought. In A. tequilana var. azul, the central spike of the rosette -containing the shoot apical meristem and folded leaves in early stages of development- is remarkably heat tolerant. We found that the most abundant protein in this organ is a 27 kDa protein. This protein was named mayahuelin to honor Mayáhuel, the agave goddess in the Aztec pantheon. LC-MS/MS analyses identified mayahuelin as a type I RIP (Ribosome Inactivating Protein). In addition to the spike, mayahuelin was expressed in the peduncle and in seeds, whereas in mature leaves, anthers, filaments, pistils, and tepals was absent. Anti-mayahuelin antibody raised against the A. tequilana var. azul protein revealed strong signals in spike leaves of A. angustifolia, A. bracteosa, A. rhodacantha, and A. vilmoriniana, and moderate signals in A. isthmensis, A. kerchovei, A. striata ssp. falcata, and A. titanota, indicating conservation at the protein level throughout the Agave genus. As in charybdin, a type I RIP characterized in Drimia maritima, mayahuelin from A. tequilana var. azul contains a natural aa substitution (Y76D) in one out of four aa comprising the active site. The RIP gene family in A. tequilana var. azul consists of at least 12 genes and Mayahuelin is the only member encoding active site substitutions. Unlike canonical plant RIPs, expression of Mayahuelin gene in S. cerevisiae did not compromise growth. The inhibitory activity of the purified protein on a wheat germ in vitro translation system was moderate. Mayahuelin orthologs from other Agave species displayed one of six alleles at Y76: (Y/Y, D/D, S/S, Y/D, Y/S, D/S) and proved to be useful markers for phylogenetic analysis. Homozygous alleles were more frequent in wild accessions whereas heterozygous alleles were more frequent in cultivars. Mayahuelin sequences from different wild populations of A. angustifolia and A. rhodacantha allowed the identification of accessions closely related to azul, manso, sigüín, mano larga, and bermejo varieties of A. tequilana and var. espadín of A. angustifolia. Four A. rhodacantha accessions and A. angustifolia var. espadín were closer relatives of A. tequilana var. azul than A. angustifolia wild accessions or other A. tequilana varieties.

Heat stress reveals high molecular mass proteasomes in Arabidopsis thaliana suspension cells cultures.

Because of their sessile nature, plants have evolved complex and robust mechanisms to respond to adverse environments. Stress conditions trigger an increase in protein turnover and degradation. Proteasomes are essential to the cell for removing, in a highly regulated manner, partially denatured or oxidized proteins thus minimizing their cytotoxicity. We observed that suspension cells of Arabidopsis thaliana treated with high temperature (37 °C) directed the assembly of high molecular mass proteasomes. The removal of a 75% of the original ubiquitin conjugates and the maintenance of protein carbonyls at basal levels correlated with a specific proteasome profiles. The profiles obtained by the separation of different proteasomes populations by Blue-Native Polyacrylamide Gel Electrophoresis and western blot analysis suggest that synthesis, assembly, and heavy ubiquitination of 20S (CP) subunits are promoted by heat stress.

Erratum to: A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis.

The original version of this article unfortunately contains a mistake. The authors have inadvertently incorrectly listed the concentration of TCA in the acetone/TCA/ß-ME solution in the materials and methods section of this paper. The TCA concentration in Sects. 2.3.2 and 2.3.5 should be 10% TCA, making the solution acetone/10% TCA/0.07% ß-ME. It is now corrected with this erratum.

A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis.

Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-ß-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.

Root hydrotropism and thigmotropism in Arabidopsis thaliana are differentially controlled by redox status.

Factors that affect the direction of root growth in response to environmental signals influence crop productivity. We analyzed the root tropic responses of thioredoxin (trxs), thigmotropic (wav2-1), and hydrotropic (ahr1 and nhr1) Arabidopsis thaliana mutants treated with low concentrations of paraquat (PQ), which induces mild oxidative stress, and established a new method for evaluating root waviness (root bending effort, RBE). This method estimates root bending by measuring and summing local curvature over the whole length of the root, regardless of the asymmetry of the wavy pattern under thigmostimulation. In roots of the wav2-1 mutant, but not in those of the trxs and ahr1 mutants, RBE was significantly inhibited under mild oxidative stress. Thigmotropic stimulation of wav2-1 mutant roots, with or without PQ treatment, showed high levels of reactive oxygen species fluorescence, in contrast to roots of the ahr1 mutant. Furthermore, PQ inhibited root growth in all genotypes tested, except in the wav2-1 mutant. In a hydrotropism assay of the trxs and wav2-1 mutants, root growth behavior was similar to the wild type with and without PQ, while the root growth of ahr1 and nhr1 mutants was diminished with PQ. These results indicate that hydrotropic and thigmotropic mutants respond differently to exogenous PQ, depending on the tropic stimulus perceived. Therefore, the mechanisms underlying hydrotropism and thigmotropism may differ.

Nodulin 22, a novel small heat-shock protein of the endoplasmic reticulum, is linked to the unfolded protein response in common bean.

The importance of plant small heat shock proteins (sHsp) in multiple cellular processes has been evidenced by their unusual abundance and diversity; however, little is known about their biological role. Here, we characterized the in vitro chaperone activity and subcellular localization of nodulin 22 of Phaseolus vulgaris (PvNod22; common bean) and explored its cellular function through a virus-induced gene silencing-based reverse genetics approach. We established that PvNod22 facilitated the refolding of a model substrate in vitro, suggesting that it acts as a molecular chaperone in the cell. Through microscopy analyses of PvNod22, we determined its localization in the endoplasmic reticulum (ER). Furthermore, we found that silencing of PvNod22 resulted in necrotic lesions in the aerial organs of P. vulgaris plants cultivated under optimal conditions and that downregulation of PvNod22 activated the ER-unfolded protein response (UPR) and cell death. We also established that PvNod22 expression in wild-type bean plants was modulated by abiotic stress but not by chemicals that trigger the UPR, indicating PvNod22 is not under UPR control. Our results suggest that the ability of PvNod22 to suppress protein aggregation contributes to the maintenance of ER homeostasis, thus preventing the induction of cell death via UPR in response to oxidative stress during plant-microbe interactions.

The Neurospora crassa DCC-1 protein, a putative histidine kinase, is required for normal sexual and asexual development and carotenogenesis.

Two-component signaling pathways based on phosphoryl group transfer between histidine kinase and response regulator proteins regulate environmental responses in bacteria, archaea, plants, slime molds, and fungi. Here we characterize a mutant form of DCC-1, a putative histidine kinase encoded by the NCU00939 gene of the filamentous fungus Neurospora crassa. We show that this protein participates in the regulation of processes such as conidiation, perithecial development, and, to a certain degree, carotenogenesis. Furthermore, DCC-1 is suggested to exert its effect by promoting cyclic AMP production, thereby placing this protein within the context of a signaling pathway.

Small heat-shock proteins and leaf cooling capacity account for the unusual heat tolerance of the central spike leaves in Agave tequilana var. Weber.

Agaves are perennial crassulacean acid metabolism (CAM) plants distributed in tropical and subtropical arid environments, features that are attractive for studying the heat-shock response. In agaves, the stress response can be analysed easily during leaf development, as they form a spirally shaped rosette, having the meristem surrounded by folded leaves in the centre (spike) and the unfolded and more mature leaves in the periphery. Here, we report that the spike of Agave tequilana is the most thermotolerant part of the rosette withstanding shocks of up to 55 degrees C. This finding was inconsistent with the patterns of heat-shock protein (Hsp) gene expression, as maximal accumulation of Hsp transcripts was at 44 degrees C in all sectors (spike, inner, middle and outer). However, levels of small HSP (sHSP)-CI and sHSP-CII proteins were conspicuously higher in spike leaves at all temperatures correlating with their thermotolerance. In addition, spike leaves showed a higher stomatal density and abated more efficiently their temperature several degrees below that of air. We propose that the greater capacity for leaf cooling during the day in response to heat stress, and the elevated levels of sHSPs, constitute part of a set of strategies that protect the SAM and folded leaves of A. tequilana from high temperatures.

Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages.

It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.

Anti-inflammatory activity of cacalol and cacalone sesquiterpenes isolated from Psacalium decompositum.

The hexane extract and two sesquiterpenic compounds, cacalol and cacalone, were isolated from the roots and rhizomes of Psacalium decompositum. Then, their anti-inflammatory activity was evaluated in carrageenan-induced rat paw edema and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema. Indomethacin was used as the anti-inflammatory agent of reference. In the rat paw model of inflammation, both the hexane extract and the sesquiterpenes isolated from Psacalium decompositum showed a clear dose-dependent inhibition of the carrageenan-induced edema (P < 0.05), with important differences among them during the temporal course of the inhibition. In the TPA-induced mouse ear edema all tested compounds showed anti-inflammatory activity in dose-dependent manner (P < 0.05). In both models, cacalone showed the most prominent anti-inflammatory activity. We conclude that some of the beneficial effects attributed to Psacalium decompositum in traditional medicine can be related with the anti-inflammatory activity of cacalol and cacalone.

[The stress response in the yeast Saccharomyces cerevisiae]. / La respuesta a estrés en la levadura Saccharomyces cerevisiae.

All living organisms are subject to changing environmental conditions, to which they must adapt in order to survive. Recently, there have been significant advances leading to the comprehension of the different mechanisms implicated in the responses to stressful situations in the yeast S. cerevisiae. In nature, as well as in laboratory conditions or industrial processes, this yeast is subjected to different adverse environmental situations, such as osmotic, thermal and oxidative stresses. A general stress response pathway, mediated by protein kinase A, allows S. cerevisiae to cope with these three stressful conditions. However, there are also specific response pathways that include the HOG kinase for osmotic stress, the Heat Shock Factor for thermal stress and Yap1p and Yap2p transcription factors that regulate the oxidative stress response, among other enzymatic and non-enzymatic mechanisms. In this review, we describe the perception and signal transduction pathways that regulate gene expression leading to the adaptation to most common types of stress in S. cerevisiae. We also include information regarding the interaction between the signal transduction pathways involved in the different responses that allow this organism to coordinate its various physiological processes for optimal adaptation to the changing environment.

Asexual development is increased in Neurospora crassa cat-3-null mutant strains.

We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3(RIP) colonies were sensitive to H(2)O(2), indicating that wild-type (Wt) resistance to external H(2)O(2) was due to CAT-3. cat-3(RIP) colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3(RIP) mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3(RIP) mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

Cu,Zn-superoxide dismutase of Saccharomyces cerevisiae is required for resistance to hyperosmosis.

Here we analyzed the role of the antioxidant response in Saccharomyces cerevisiae adaptation to hyperosmotic stress. We show that Cu,Zn-superoxide dismutase (SOD1) plays a fundamental role in this adaptation process since under hyperosmosis SOD1 mutants lead to high protein oxidation levels and show a sensitive phenotype, which is reversed by the addition of N-acetylcysteine to the medium. Pretreatment with MnCl(2), a superoxide scavenger, improves the survival of the sod1 strain upon hyperosmosis. Additionally, we show that upon hyperosmotic shock there is a small and transient increase in SOD1 transcript levels, regulated by the protein kinase A-cAMP and SKN7 pathways.

Regulation and oxidation of two large monofunctional catalases.

The two Neurospora crassa catalase genes cat-1 and cat-3 were shown to encode Cat-1 and Cat-3 large monofunctional catalases. cat-1 and cat-3 genes are regulated differentially during the asexual life cycle and under stress conditions. A stepwise increase in catalase activity occurs during conidiation. Conidia have 60 times more catalase activity than exponentially growing hyphae. Cat-1 activity was predominant in conidia, during germination and early exponential growth. It was induced during prestationary growth and by ethanol or heat shock. Cat-3 activity was predominant during late exponential growth and at the start of the conidiation process. It was induced under stress conditions, such as H(2)O(2), paraquat, cadmium, heat shock, uric acid, and nitrate treatment. In general, Cat-1 activity was associated with nongrowing cells and Cat-3 activity with growing cells. The Cat-3 N-terminus sequence indicates that this catalase is processed and presumably secreted. Paraquat caused modification and degradation of Cat-1. Under heat shock both Cat-1 and Cat-3 were modified and degraded and Cat-1 was resynthesized. Paraquat and heat shock effects were observed only in the presence of air and are probably related to in vivo generation of singlet oxygen. Purified Cat-3 was modified with a photosensitizing reaction in which singlet oxygen is produced.