Deciphering fruit sugar transport and metabolism from tolerant and sensitive tomato plants subjected to simulated field conditions

In the current state of climate change, we must assume that abiotic stresses act together under natural field conditions, these will increase in the coming years. Therefore, in this report we investigated how sugar metabolism was affected under simulated field conditions, where plants faced high ambient temperatures and a low-quality water irrigation. Our studies were carried out on fruits of two tomato recombinant lines, a tolerant and a sensitive one exposed to the combination of heat and salinity. Two ripening stages (mature green and red ripe fruits) were used in our analyzes, where the gene expression levels of the main biosynthetic genes and transporters, enzymatic activities and compounds related to the synthesis, accumulation, and degradation of sugars in plants were analyzed. The tolerant line showed highly significant differences in red ripe fruits in comparison to the sensitive one under the simulated field conditions (35°C + 60 mM NaCl), with an overexpression of the genes SlFBP, SlSPS, SlSUS3, and SlNi. These expression patterns correlated with a higher activity of the enzymes FBP, SPS, SUS3, AI, and G6PDH, which resulted in the accumulation of fructose, glucose and UDP-glucose. Our results showed the advantage of using tomato recombinant lines for rescuing important traits, such as the resistance to some abiotic stresses, and for the identification of important molecular and metabolic markers that could be used to determine fruit quality in green or red maturity stages under detrimental environmental field conditions.Ministry of Economy and Competitiveness from Spain, Grant/Award Numbers: PGC2018-095731-B-I00, AGL2015-66033-R; Ministry of Science, Innovation and Universities of Spain, Grant/Award Number: FPU16/0526

CP-154,526 Modifies CREB Phosphorylation and Thioredoxin-1 Expression in the Dentate Gyrus following Morphine-Induced Conditioned Place Preference.

Corticotropin-releasing factor (CRF) acts as neuro-regulator of the behavioral and emotional integration of environmental and endogenous stimuli associated with drug dependence. Thioredoxin-1 (Trx-1) is a functional protein controlling the redox status of several proteins, which is involved in addictive processes. In the present study, we have evaluated the role of CRF1 receptor (CRF1R) in the rewarding properties of morphine by using the conditioned place preference (CPP) paradigm. We also investigate the effects of the CRF1R antagonist, CP-154,526, on the morphine CPP-induced activation of CRF neurons, CREB phosphorylation and Trx expression in paraventricular nucleus (PVN) and dentate gyrus (DG) of the mice brain. CP-154,526 abolished the acquisition of morphine CPP and the increase of CRF/pCREB positive neurons in PVN. Moreover, this CRF1R antagonist prevented morphine-induced CRF-immunoreactive fibers in DG, as well as the increase in pCREB expression in both the PVN and DG. In addition, morphine exposure induced an increase in Trx-1 expression in DG without any alterations in PVN. We also observed that the majority of pCREB positive neurons in DG co-expressed Trx-1, suggesting that Trx-1 could activate CREB in the DG, a brain region involved in memory consolidation. Altogether, these results support the idea that CRF1R antagonist blocked Trx-1 expression and pCREB/Trx-1 co-localization, indicating a critical role of CRF, through CRF1R, in molecular changes involved in morphine associated behaviors

Using Tomato Recombinant Lines to Improve Plant Tolerance to Stress Combination Through a More Efficient Nitrogen Metabolism

The development of plant varieties with a better nitrogen use efficiency (NUE) is a means for modern agriculture to decrease environmental pollution due to an excess of nitrate and to maintain a sufficient net income. However, the optimum environmental conditions for agriculture will tend to be more adverse in the coming years, with increases in temperatures, water scarcity, and salinity being the most important productivity constrains for plants. NUE is inherently a complex trait, as each step, including N uptake, translocation, assimilation, and remobilization, is governed by multiple interacting genetic and environmental factors. In this study, two recombinant inbred lines (RIL-66 and RIL-76) from a cross between Solanum lycopersicum and Solanum pimpinellifoilum with different degree of tolerance to the combination of salinity and heat were subjected to a physiological, ionomic, amino acid profile, and gene expression study to better understand how nitrogen metabolism is affected in tolerant plants as compared to sensitive ones. The ionomics results showed a different profile between the two RILs, with K+ and Mg2+ being significantly lower in RIL-66 (low tolerant) as compared to RIL-76 (high tolerant) under salinity and heat combination. No differences were shown between the two RILs in N total content; however, N-NO3− was significantly higher in RIL-66, whereas N-Norg was lower as compared to the other genotype, which could be correlated with its tolerance to the combination of salinity and heat. Total proteins and total amino acid concentration were significantly higher in RIL-76 as compared to the sensitive recombinant line under these conditions. Glutamate, but more importantly glutamine, was also highly synthesized and accumulated in RIL-76 under the combination of salinity and heat, which was in agreement with the upregulation of the nitrogen metabolism related transcripts studied (SlNR, SlNiR, SlGDH, SlGLT1, SlNRT1.2, SlAMT1, and SlAMT2). This study emphasized the importance of studying abiotic stress in combination and how recombinant material with different degrees of tolerance can be highly important for the improvement of nitrogen use efficiency in horticultural plants through the targeting of N-related markers.This work was supported by the Ministry of Economy and Competitiveness from Spain (Grant No. AGL2015-66033-R and Grant No. PGC2018-095731-B-I00, MCIU/AEI/FEDER, UE

Experimental schedule for the conditioned place preference training.

<p>Mice were pre-treated with CP-154,526 (30 mg/kg, i.p.) or vehicle (Tween 80) 30 min before saline or morphine for 6 days in the conditioning period.</p

The analyzed region within dentate gyrus (DG) is schematically illustrated in A (diagram from Franklin & Paxinos 2008); coordinate -1.94 mm from Bregma.

<p>(B) Example of magnification micrograph showing a midbrain coronal section of mice immunostained for pCREB and Trx-1; scale bar 100 μm. Representative confocal images of pCREB (red) (C-F) and Trx-1 (green) (C’-F’). Merged images are shown in C”-F” (pCREB/Trx-1). Colocalization is shown by yellow-orange neurons in the merged images. Scale bars 20 μm. ***<i>p</i><0.001, versus vehicle (veh)+saline (S); +<i>p</i><0.05, ++<i>p</i><0.01 versus vehicle+morphine (M). CP-154,526 (CP).</p

CREB activation in morphine CPP mice.

<p>Photographs represent immunohistochemical detection of pCREB in paraventricular nucleus (PVN) (A), and dentate gyrus (DG) (C). Scale bar 100 μm. Quantitative analysis of pCREB immunohistochemistry in PVN (B) and DG (D). Data are expressed as mean ± SEM. **<i>p</i><0.01, ***<i>p<</i>0.001 versus vehicle (veh)+ saline (S); +<i>p</i><0.05, +++<i>p</i><0.001 versus vehicle+morphine (M). CP-154,526 (CP).</p

Example of photomicrographs showing CRF/pCREB double-labelling in paraventricular nucleus (PVN) (A).

<p>Graph in upper right shows the mean total number or double-labeled neurons (B). Photographs represent immunohistochemical detection of CRF fibers in the dentate gyrus (DG) (C). Graph in downright shows the optical density of CRF immunostaining in the DG (D). Scale bar 100 or 50 μm. Data are expressed as mean ± SEM. ***<i>p</i><0.001 versus vehicle (veh)+saline (S); ++<i>p</i><0.01, +++p<0.001 versus vehicle+morphine (M). CP-154,526 (CP).</p

Western-blotting analysis of Trx-1 in paraventricular nucleus (PVN) (A) and dentate gyrus (DG) (B).

<p>Each bar represents the mean optical density ± SEM; values are expressed as % of controls.*<i>p</i><0.05 versus vehicle+saline; ++<i>p</i><0.01 versus vehicle+morphine.</p

Effects of CP-154,526 on weight gain in mice treated with morphine or saline, n = 11–13 (A).

<p>Effect of repeated saline or morphine injection on conditioned place preference, (CPP). CPP induced by morphine administration (6 mg/kg, i.p.) during the conditioning phase (B). The score was calculated for each mouse as the difference between post-conditioning and the preconditioning time spent in the drug-paired compartment. Data are expressed as mean ± SEM. **<i>p</i><0.01, ***<i>p</i><0.001 versus the control group treated with vehicle+saline; &&<i>p</i><0.01, &&&<i>p</i><0.001 versus the group treated with CP-154,526+saline; +++<i>p</i><0.001 versus the group treated with vehicle+morphine.</p

The analyzed region within paraventricular nucleus (PVN) is schematically illustrated in A (diagram from Franklin & Paxinos 2008); coordinate -0.82 mm from Bregma.

<p>(B) Example of magnification micrograph showing a midbrain coronal section of mice immunostained for pCREB and Trx-1; scale bar 100 μm. Representative confocal images of pCREB (reed) (C-F) and Trx-1 (green) (C’-F’). Merged images are shown in C”-F” (pCREB/Trx-1). Scale bars 20 μm. Graphs in right show the mean total number of pCREB (G) or Trx-1 (H) neurons and the double-labelled neurons (pCREB/Trx-1) (I). Data are expressed as mean ± SEM. **<i>p</i><0.01, versus vehicle (veh)+ saline (S); ++<i>p</i><0.01 versus vehicle+morphine (M). CP-154,526 (CP).</p