Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica)

[EN] Background Salt stress is one of the main constraints determining crop productivity, and therefore one of the main limitations for food production. The aim of this study was to characterize the salt stress response at the physiological and molecular level of different Broccoli (Brassica oleracea L. var. Italica Plenck) cultivars that were previously characterized in field and greenhouse trials as salt sensitive or salt tolerant. This study aimed to identify functional and molecular traits capable of predicting the ability of uncharacterized lines to cope with salt stress. For this purpose, this study measured different physiological parameters, hormones and metabolites under control and salt stress conditions. Results This study found significant differences among cultivars for stomatal conductance, transpiration, methionine, proline, threonine, abscisic acid, jasmonic acid and indolacetic acid. Salt tolerant cultivars were shown to accumulate less sodium and potassium in leaves and have a lower sodium to potassium ratio under salt stress. Analysis of primary metabolites indicated that salt tolerant cultivars have higher concentrations of several intermediates of the Krebs cycle and the substrates of some anaplerotic reactions. Conclusions This study has found that the energetic status of the plant, the sodium extrusion and the proline content are the limiting factors for broccoli tolerance to salt stress. Our results establish physiological and molecular traits useful as distinctive markers to predict salt tolerance in Broccoli or to design novel biotechnological or breeding strategies for improving broccoli tolerance to salt stress.This work was funded by Grant RTC-2017-6468-2-AR (APROXIMACIONES MOLECULARES PARA INCREMENTAR LA TOLERANCIA A SALINIDAD Y SEQUiA DEL BROCOLI) funded by MCIN/AEI/10.13039/501100011033 and by "ERDF A way of making Europe" by the European Union. S.C. is a recipient of grant FPU19/01977 from the Spanish Ministerio de Universidades. L.M. was supported by the Spanish MICINN (PTA2019-018094). L.M and A.V. activities were founded by Prometeu program (IMAGINA project, PROMETEU/2019/110). CEAM foundation is funded by Generalitat Valenciana. None of the funding bodies has participated in the design of the study or the collection, analysis, interpretation of data, nor in writing the manuscript.Chevilly-Tena, S.; Dolz-Edo, L.; Morcillo, L.; Vilagrosa, A.; López-Nicolás, JM.; Yenush, L.; Mulet, JM. (2021). Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica). BMC Plant Biology. 21(1):1-16. https://doi.org/10.1186/s12870-021-03263-4S11621

Deciphering dynamic dose responses of natural promoters and single cis elements upon osmotic and oxidative stress in yeast

[EN] Fine-tuned activation of gene expression in response to stress is the result of dynamic interactions of transcription factors with specific promoter binding sites. In the study described here we used a time-resolved luciferase reporter assay in living Saccharomyces cerevisiae yeast cells to gain insights into how osmotic and oxidative stress signals modulate gene expression in a dose-sensitive manner. Specifically, the dose-response behavior of four different natural promoters (GRE2, CTT1, SOD2, and CCP1) reveals differences in their sensitivity and dynamics in response to different salt and oxidative stimuli. Characteristic dose-response profiles were also obtained for artificial promoters driven by only one type of stress-regulated consensus element, such as the cyclic AMP-responsive element, stress response element, or AP-1 site. Oxidative and osmotic stress signals activate these elements separately and with different sensitivities through different signaling molecules. Combination of stress-activated cis elements does not, in general, enhance the absolute expression levels; however, specific combinations can increase the inducibility of the promoter in response to different stress doses. Finally, we show that the stress tolerance of the cell critically modulates the dynamics of its transcriptional response in the case of oxidative stress.This work was supported by the Ministerio de Economa y Competitividad (grant BFU2011-23326 to M.P.) and the Ministerio de Ciencia e Innovacion (predoctoral FPI grant to A.R.).Dolz Edo, L.; Rienzo, A.; Poveda Huertes, D.; Pascual-Ahuir Giner, MD.; Proft, MH. (2013). Deciphering dynamic dose responses of natural promoters and single cis elements upon osmotic and oxidative stress in yeast. Molecular and Cellular Biology. 33(11):2228-2240. https://doi.org/10.1128/MCB.00240-13222822403311Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., … Brown, P. O. (2000). 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Distinctive Traits for Drought and Salt Stress Tolerance in Melon (Cucumis melo L.)

Melon (Cucumis melo L.) is a crop with important agronomic interest worldwide. Because of the increase of drought and salinity in many cultivation areas as a result of anthropogenic global warming, the obtention of varieties tolerant to these conditions is a major objective for agronomical improvement. The identification of the limiting factors for stress tolerance could help to define the objectives and the traits which could be improved by classical breeding or other techniques. With this objective, we have characterized, at the physiological and biochemical levels, two different cultivars (sensitive or tolerant) of two different melon varieties (Galia and Piel de Sapo) under controlled drought or salt stress. We have performed physiological measurements, a complete amino acid profile and we have determined the sodium, potassium and hormone concentrations. This has allowed us to determine that the distinctive general trait for salt tolerance in melon are the levels of phenylalanine, histidine, proline and the Na+/K+ ratio, while the distinctive traits for drought tolerance are the hydric potential, isoleucine, glycine, phenylalanine, tryptophan, serine, and asparagine. These could be useful markers for breeding strategies or to predict which varieties are likely perform better under drought or salt stress. Our study has also allowed us to identify which metabolites and physiological traits are differentially regulated upon salt and drought stress between different varieties.SC was a recipient of grant FPU19/01977 from the Spanish Ministerio de Universidades. LM and AV activities were funded by the Prometeu program (IMAGINA project, PROMETEU/2019/110). LM was also supported by the Spanish MICINN (PTA2019-018094). The CEAM foundation was funded by the Generalitat Valenciana

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability

The evolutionary conserved TOR complex 1 controls growth in response to the quality and quantity of nutrients such as carbon and amino acids. The protein kinase Sch9 is the main TORC1 effector in yeast. However, only few of its direct targets are known. In this study, we performed a genome-wide screening looking for mutants which require Sch9 function for their survival and growth. In this way, we identified multiple components of the highly conserved vacuolar proton pump (V-ATPase) which mediates the luminal acidification of multiple biosynthetic and endocytic organelles. Besides a genetic interaction, we found Sch9 also physically interacts with the V- ATPase to regulate its assembly state in response to glucose availability and TORC1 activity. Moreover, the interaction with the V-ATPase has consequences for ageing as it allowed Sch9 to control vacuolar pH and thereby trigger either lifespan extension or lifespan shortening. Hence, our results provide insights into the signaling mechanism coupling glucose availability, TORC1 signaling, pH homeostasis and longevity. As both Sch9 and the V-ATPase are highly conserved and implicated in various pathologies, these results offer fertile ground for further research in higher eukaryotes

Identification of Distinctive Primary Metabolites Influencing Broccoli (<i>Brassica oleracea</i>, var. <i>Italica</i>) Taste

Broccoli (Brassica oleracea L. var. Italica Plenck) is a cruciferous crop that is considered to be a good source of micronutrients. Better taste is a main objective for breeding, as consumers are demanding novel cultivars suited for a healthy diet, but ones that are more palatable. This study aimed to identify primary metabolites related to cultivars with better taste according to a consumer panel. For this purpose, we performed a complete primary metabolomic profile of 20 different broccoli cultivars grown in the field and contrasted the obtained data with the results of a consumer panel which evaluated the taste of the same raw buds. A statistical analysis was conducted to find primary metabolites correlating with better score in the taste panels. According to our results, sugar content is not a distinctive factor for taste in broccoli. The accumulation of the amino acids leucine, lysine and alanine, together with Myo-inositol, negatively affected taste, while a high content of γ-aminobutyric acid (GABA) is a distinctive trait for cultivars scoring high in the consumer panels. A Principal Component Analysis (PCA) allowed us to define three different groups according to the metabolomic profile of the 20 broccoli cultivars studied. Our results suggest molecular traits that could be useful as distinctive markers to predict better taste in broccoli or to design novel biotechnological or classical breeding strategies for improving broccoli taste

Physiological and Molecular Characterization of the Differential Response of Broccoli (Brassica oleracea var. Italica) Cultivars Reveals Limiting Factors for Broccoli Tolerance to Drought Stress

Broccoli is a cruciferous crop rich in health-promoting metabolites. Due to several factors, including anthropogenic global warming, aridity is increasing in many cultivation areas. There is a great demand to characterize the drought response of broccoli and use this knowledge to develop new cultivars able to maintain yield under water constraints. The aim of this study is to characterize the drought response at the physiological and molecular level of different broccoli (Brassica oleracea L. var. Italica Plenck) cultivars, previously characterized as drought-sensitive or drought-tolerant. This approach aims to identify different traits, which can constitute limiting factors for drought stress tolerance in broccoli. For this purpose, we have compared several physiological parameters and the complete profiles of amino acids, primary metabolites, hormones, and ions of drought-tolerant and drought-sensitive cultivars under stress and control conditions. We have found that drought-tolerant cultivars presented higher levels of methionine and abscisic acid and lower amounts of urea, quinic acid, and the gluconic acid lactone. Interestingly, we have also found that a drought treatment increases the levels of most essential amino acids in leaves and in florets. Our results have established physiological and molecular traits useful as distinctive markers to predict drought tolerance in broccoli or which could be reliably used for breeding new cultivars adapted to water scarcity. We have also found that a drought treatment increases the content of essential amino acids in broccoli.The authors thank Prof. Angel Maquieira for generously providing the technical equipment required for ion determination. S.C.’s work was funded by Grant FPU19/01977 from the Spanish Ministerio de Universidades. This work was funded by the RTC-2017-6468-2-AR project (APROXIMACIONES MOLECULARES PARA INCREMENTAR LA TOLERANCIA A SALINIDAD Y SEQUíA DEL BRÓCOLI) awarded by the “Agencia estatal de Investigación”. L.M. was supported by the Spanish MICINN (PTA2019-018094). Activities of L.M. and A.V. were funded by the Prometeu program (IMAGINA project, PROMETEU/2019/110). The CEAM foundation is funded by Generalitat Valenciana. The authors thank SAKATA for the generous gift of plant material

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability

The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.status: publishe

Function of Sch9 in regulating ageing is dependent on V-ATPase activity.

<p>With the exception of panel E, all chronological ageing data represent measurements performed on cells at day 8 in stationary phase. (A) Cell survival and (B) ROS determination of strains aged in non-buffered fully supplemented medium as determined by flow cytometry. (C) Cell survival and (D) ROS levels of strains aged in fully supplemented medium buffered at pH 5.5 as determined by flow cytometry. (E) Cell survival of strains grown in buffered medium at day 23 in stationary phase as determined by CFU counting. (F) pH of the culture medium of ageing cells grown in buffered medium. (G) Cell survival and (H) ROS determination of strains grown in medium containing the indicated concentration of methionine as determined by flow cytometry. Results depicted are mean values ± SD. (I) Sch9 affects pHv. Vacuolar pH was measured during exponential growth and during glucose starvation using the ratiometric fluorescent pH indicator BCECF-AM. Results depicted are mean values ± SEM of four independent experiments. All differences between strains and conditions are statistically significant unless stated as ns (not significant). A detailed statistical analysis is presented in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s013" target="_blank">S3 Table</a></b>. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s007" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s008" target="_blank">S8</a> Figs</b>.</p

Effects on colony size and growth by deletion of <i>VPH1</i>, <i>STV1</i> and/or <i>SCH9</i>.

<p>(A, B) A synthetic sick phenotype arises when deletion of <i>SCH9</i> is combined with a fully dysfunctional V-ATPase. (A) Tetrad dissection of the diploid strain JW 04 952 (<i>sch9Δ/SCH9 vph1Δ/VPH1 stv1Δ/STV1</i>). (B) Colony sizes were calculated, normalized relative to WT and are shown as mean values ± SD. Letters indicate groups of strains with a significant difference in colony size (p < 0.001, one-way ANOVA). (C, D) Strains combining deletion of <i>SCH9</i> with a fully dysfunctional V-ATPase show a deteriorated growth phenotype. OD_600nm was followed over time in fully supplemented medium without buffer (C) or buffered at pH 5 (D). A representative experiment with at least 4 independent colonies for each strain is shown. Error bars represent SD from the mean. See also <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s004" target="_blank">S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006835#pgen.1006835.s005" target="_blank">S5</a> Figs</b>.</p

Phenotypic overlap between <i>sch9Δ</i> and mutants in which V-ATPase function is impaired.

<p>Phenotypic overlap between <i>sch9Δ</i> and mutants in which V-ATPase function is impaired.</p