Rubén D. Conde

Accumulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase RNA under biological stress conditions and elicitor treatments in potato

Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whether the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. This increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts sinergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions.

Trichoderma spp. as elicitors of wheat plant defense responses against Septoria tritici

Abstract Leaf blotch of wheat is a widespread and highly active disease that affects wheat production. In addition to the use of chemicals and proper cultivation methods, microbial antagonists are used to control plant pathogens. Trichoderma spp. stimulate a systemic induced response in plants. Therefore, the efficacy of Trichoderma spp. against wheat leaf blotch was evaluated under greenhouse conditions. The susceptible plants were sprayed with Septoria tritici conidiospores. In order to select an efficient method of pretreatment with Trichoderma spp., leaf spraying and seed coating with 14 isolates were tested in 2003 and 2004. The extent of leaf necrosis area and pycnidial coverage was estimated. Antagonism was assessed by the capacity of each Trichoderma spp. isolate to restrict the progress of leaf blotch, 21 days after inoculation. Of the two methods, seed coating was more efficacious against leaf blotch than leaf spraying. Amongst the 14 isolates tested, the isolate prepared from T. harzianum (Th5) produced the highest level of protection. None of the treatments caused changes in plant stem diameter or dry weight. Trichoderma spp. did not get into leaves while S. tritici was present, even in asymptomatic leaf extracts. In addition, the leaf apoplast antifungal proteolytic activity was measured in plants 7, 15, and 22 days after sowing. This antifungal action decreased in plants only inoculated with S. tritici, but increased in those grown from seeds coated with the T. harzianum (Th5) isolate. This increase conferred resistance to the susceptible wheat cultivar. The endogenous germin-like protease inhibitor coordinated the proteolytic action. These results suggest that T. harzianum stimulates a biochemical systemic induced response against leaf blotch.

Dietary level of protein regulates glyceraldehyde-3-phosphate dehydrogenase content and synthesis rate in mouse liver cytosol

The content of liver cytosolic proteins was studied in mice subjected to protein depletion followed by refeeding with a normal diet. Depletion elicited either the accumulation or the decrease of several polypeptides, being the early increase of a Mr 36 000 polypeptide the most pronounced change observed. The refeeding with a normal diet for 2 days caused a return of the cytosol protein composition to that of normally fed animals. The Mr 36 000 polypeptide was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Its molecular weight, the sequence of its first twenty amino acid residues, its amino acid composition and its antigenic properties were found to be similar with those of GAPDH from different mammalian cells. During the first 2 days of protein depletion, both the GAPDH polypeptide content and activity increased. Thereafter, the enzymatic activity of GAPDH decreased, whereas GAPDH protein mass decreased in a lesser extent. The accumulation of GAPDH and other particular polypeptides in the cytosols of protein depleted mice was associated with an increased synthesis. The refeeding with a normal diet caused an immediate return to the synthesis pattern of normal livers.

Two dimensional non-equilibrium pH gel electrophoresis mapping of cytosolic protein changes caused by dietary protein depletion in mouse liver

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Yb1, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or comparison of two-dimensional gel electrophoretic patterns, carbonic anhydrase III (CAIII), Cu, Zn superoxide dismutase (CuZnSOD) and a cytochrome P450 cytosolic protein (cyt P450) were identified. These three proteins, as well as GSTs, are related with intracellular detoxification and free radical scavenging systems. Their contents were regulated by dietary protein restriction in a manner indicative of diminished liver defence against oxidising agents.

Contribution of breakdown in the regulation of mouse liver glutathione S-transferase subunits during protein depletion and re-feeding.

The in vivo radioactivity decay of glutathione S-transferase (GST) was observed in livers of normal-fed (N), protein-depleted (D), and re-fed mice (R), labelled with [35S]methionine. Half-lives in days at N, D and R, respectively, were: total GST, 1.7, 1.4, 4.3; Yb1-subunit, 2.0, 1.8, 3.3; Yc-subunit, 2.4, 1.0, 4.2; Yf-subunit, 1. 1, 2.1, 5.0. These findings, together with the fact that the proportions of GST-subunits depend on dietary protein, show that breakdown contributes differentially to control the content of each GST subunit. Data also indicate that GSTs are long-life proteins.

Estimation of ubiquitin and ubiquitin mRNA content in dark senescing wheat leaves

Wheat leaves(Triticum aestivum L. cv. San Agustin INTA) were detached at the moment they had reached maximum expansion, put in tubes containing water and left in darkness. Under these conditions, leaf protein content decreased mainly as consequence of an increased rate of breakdown. In the range of 0 to 72 h after detachment, western blot analysis of leaf protein extracts displayed both similar proportions of total protein and quality of ubiquitin conjugates. Northern blot analysis of leaf RNA extracts revealed a 1.6 kb ubiquitin mRNA transcript which increased 3.5-fold after 48 h of treatment. Thus wheat leaves maintain both their ability for the ubiquitination of proteins and the transcription of ubiquitin mRNA at stages of senescence in which rates of protein breakdown are increased. The results suggest that the ubiquitin-dependent proteolytic pathway contributes to leaf protein breakdown during senescence

Protein depletion and refeeding change the proportion of mouse liver glutathione S-transferase subunits

The effect of protein depletion followed by refeeding with a normal diet on the content of mouse liver cytosolic proteins was studied. By peptide-mass fingerprinting and N-terminal sequencing, three polypeptides whose contents changed with dietary protein level were identified as glutathione S-transferases (GST) Yb1, Yc and Yf subunits. Five days of depletion caused the increase of Yb1 and Yf (21.6% and 78.5%, respectively) and the decrease of Yc (31.2%). After two days of refeeding, Yb1 and Yc were practically restored, while the neoplastic marker Yf remained higher (63.4%). None of the nutritional conditions tested induced new GSTs. While protein depletion-refeeding altered the ratios between the constitutive GST subunits, total liver GST content and activity were unaffected by depletion and slightly increased by refeeding. The increased amounts of Yb1 and Yf, and the maintenance of total GST content, indicate that during protein depletion, the GST subunits levels are controlled by mechanisms different from the majority of cytosolic proteins.

Influence of protein nutrition on calpain activity of mouse kidney: Modulation by calpastatin

The effect of protein depletion and refeeding with a normal diet on calpain activity was examined in mouse kidney soluble homogenate. In terms of units per gram of protein, it increased 2.9 times with depletion and decreased upon refeeding. After a DEAE-Sephacel chromatography, the homogenate yielded three enzymatic activities. Their sum, assessed as total calpain activity, was higher than the activity measured before fractionation and did not appreciably change during protein depletion and refeeding. Because the proportion of total activity displayed by the complete homogenate increased with depletion and decreased with refeeding, a low calpastatin content in depleted kidney was envisaged. This was confirmed by direct estimations: depleted kidney had 6 times less calpastatin compared to both normal and 16 h refed tissue. We concluded that a decrease in calpastatin content contributes to an increased calpain activity related to degradable protein in protein depleted kidney. In view of this, it seems not unlikely that the in vivo rate of protein breakdown depicted by kidney during protein depletion and refeeding is in part effected through modulation of the calpain proteolytic system. (Mol Cell Biochem 166: 95-99, 1997)

Protein turnover during growth of the haloalkaliphilic archaeon Natronococcus occultus

Changes in protein turnover during growth and stationary phase of Natronococcus occultus were studied. The cell capacity for protein synthesis was maximal at the exponential growth phase. This was coincident with increases in both protein/DNA an RNA/DNA ratios. At the onset of exponential growth phase, the synthesis of extracellular protein was higher than that of intracellular protein. Amino acid analysis revealed cells to contain high levels of free glutamic acid (68 to 35 fold higher than free proline), the content of which and total amino groups decreased with growth. Protein breakdown was assessed from the decay of radioactive label. At any growth phase, the half‐life of N. occultus bulk protein was shorter than those displayed by eubacteria. Because half‐life remained unchanged during lag and early exponential growth phase, the associated increase of cellular protein was ascribed to an increased synthesis. Amino acid composition analysis of bulk protein showed a strong correlation between short half‐life and high contents of hydrophobic and acidic amino acids.