The higher alcohols 2-phenylethanol, tryptophol, and tyrosol are a group of yeast-derived compounds that have been shown to affect the aroma and flavour of fermented beverages. Five variants of the industrial wine strain AWRI796, previously isolated due to their elevated production of the ‘rose-like aroma’ compound 2-phenylethanol, were characterised during pilot-scale fermentation of a Chardonnay juice. We show that these variants not only increase the concentration of 2-phenylethanol but also modulate the formation of the higher alcohols tryptophol, tyrosol, and methionol, as well as other volatile sulfur compounds derived from methionine, highlighting the connections between yeast nitrogen and sulfur metabolism during fermentation. We also investigate the development of these compounds during wine storage, focusing on the sulfonation of tryptophol. Finally, the sensory properties of wines produced using these strains were quantified at two time points, unravelling differences produced by biologically modulating higher alcohols and the dynamic changes in wine flavour over aging. Saccharomyces cerevisiae performs a wide range of industrial fermentations that functionally depend upon its ability to convert sugars to ethanol and carbon dioxide efficiently. While performing this primary function, S. cerevisiae also produces a range of secondary metabolites, such as esters, volatile fatty acids, higher alcohols, and volatile sulfur compounds (VSCs), which contribute substantially to the flavour and aroma of wine [ of wine , beer [2 of wine . Of thes of wine .Higher alcohols, also known as fusel alcohols, are compounds with more than two carbon atoms. These alcohols are derived from yeast amino acid metabolism via the Ehrlich pathway . Amino a2), which is widely used in winemaking as an additive due to its antimicrobial and antioxidant effects, to yield the tryptophol-2-sulfonate (TOL-SO3H) adduct [3H from TOL seems to be increased by bottle aging, particularly for white wines [3H on the taste and mouthfeel properties of fermented beverages remains unclear, it has recently been linked with bitterness [Except for 2-phenylethanol (2-PE), which is derived from phenylalanine and associated with a ‘rose-like’ odour quality , the ind) adduct . This re) adduct , and thete wines . Althougtterness .Higher alcohols are substrates for acetate ester production, a reaction catalysed by yeast alcohol acetyltransferases. Many acetate esters are associated with ’fruity’ and ‘floral’ aromas in wine . For exaS. cerevisiae strains that improve the organoleptic properties of sake, wine, and bread [Due to the ability of 2-PE and 2-PEA to impart floral notes, these compounds present an opportunity to shape wine style. The ability to influence the production of 2-PE and 2-PEA in white wines would be beneficial because, although naturally occurring, they are usually present at relatively low concentrations and are unlikely to impart a definitive character ,21. The nd bread ,24,25.2S). Similarly, a range of compounds derived from methionine by either enzymatic or non-enzymatic reactions are also associated with negative attributes in wine, such as methanethiol (MeSH) (‘sewage and rubber’ aromas), methional (‘cooked potato’ aroma) and methyl thioacetate (MeSAc) (‘sulfurous and cheesy’ aromas) [2S by yeast has been extensively studied, and commercial low H2S producing strains have been generated [Conversely, and with the exception of a small group of polyfunctional thiols associated with ‘fruity’ and ‘tropical’ characters , most VS aromas) ,8,27. Whenerated , little 3H and TOL during wine ageing are also reported. Formal sensory analysis was conducted on the Chardonnay wines produced by each strain at two time points, and the links between the resulting changes to chemical composition and sensory properties are presented and explored.This paper explores the influence of compounds derived from the metabolism of both aromatic and sulfur-containing amino acids in the sensory profile of Chardonnay wine over time. For this purpose, we characterised five variants of a commercially available wine yeast strain (AWRI796), which were previously isolated because of their high 2-PE production phenotype. Here we show that these variants influence not only the production of 2-PE and 2-PEA but also the concentration of the higher alcohols TOL, TyrOH, and methionol, as well as other odoriferous VSCs derived from the amino acid methionine. The progression of these compounds and the equilibrium shift between TOL-SOPreviously, we isolated a range of variants from the commercial wine yeast AWRI796 that were resistant to toxic fluorinated analogues of phenylalanine. These variants were shown to overproduce 2-PE and 2-PEA to different extents in laboratory-scale fermentations . A singlp < 0.0001, The effects of variations in 2-PE, TyrOH and TOL concentration on wine sensory properties were determined using five mutants of AWRI796 together with parent strain (control) to produce Chardonnay wine. The variant strains harbour distinct mutations in two of the enzymes involved in aromatic amino acid metabolism: Tyr1p and Aro4p , while an exponential model was a better fit when all strains were included (R2 = 0.97)  . In cont = 0.97) . While t = 0.97) . Notably3H adduct. At the end of alcoholic fermentation, only wines made with the highest TOL producer (AWRI2940) contained TOL-SO3H (0.26 mg L−1), representing a 0.3% molar conversion of TOL into TOL-SO3H. The post-fermentation concentration of total SO2 in the wines averaged 21 mg L−1, with no detectable free SO2 . Before bottling, free SO2 concentration was adjusted to between 35 and 40 mg L−1 (−1 TOL) the yield of TOL-SO3H was at least 80%, while a 26% yield was observed in wines made using the high TOL producer (AWRI2940)  .3H species was reached, with longer storage times (12 and 15 months) having little or no effect on the conversion of the higher alcohol into the sulfonated adduct   the yield was only 36%  . For theonly 36% .3H formation was a significant contributor to SO2 loss during bottle storage. On the day of bottling, the concentration of free SO2 averaged 39 mg L−1 across all wines  than those made with the parent strain (AWRI796), whereas the low 2-PE producer and the high TyrOH-producing strains  accumulated similar concentrations of methionol to the parent . The con2S was observed at any time point  while showing a slightly elevated concentration of dimethylsulfide (DMS). No statistical evidence supporting differences between the strains in the concentration of Hme point .To further confirm the effects that mutations in either Tyr1p or Aro4p might have on methionine catabolism, two of the variants were used to ferment a synthetic grape medium (SGM) with grape-like concentrations of methionine . The twop < 0.05) supports differences between strains in three attributes rated by the panel: ‘yellow colour intensity’, ‘floral aroma’ and ‘grassy flavour’ (p < 0.15) alluded to possible trends among the strains for the attributes ‘grassy aroma’, ‘cooked vegetable/potato aroma’, ‘sweetness’, ‘bitterness’, ‘stone fruit flavour’ and ‘flint flavour’. In particular, wines made with AWRI796 and AWRI4124 were rated highly in the attributes ‘cooked vegetable/potato aroma’ and ‘sweetness’, while there was a trend for ‘bitterness’ to be rated lowest for the low TOL-/TOL-SO3H-producing strains AWRI2936 and AWRI796 compared to the other strains.Sensory descriptive analysis of the wines was performed twice, after 3 and 15 months in-bottle. The analyses were compared to assess the effect of bottle storage on wine sensory attributes. The mean scores for a subset of sensory attributes are summarised in flavour’ . Wines fp < 0.001) that strains produced wines with different intensities of ‘yellow colour’, ‘cooked vegetable/potato aroma’, ‘sourness’ and ‘sweetness’. There was evidence (p < 0.05) for differences in ‘pungency’, ‘rose aroma’ and ‘stone fruit flavour’. Notably, at this time point, judges chose to rate a specific ‘rose aroma’ quality rather than the more general ‘floral’ aroma attribute used to describe the wines at three months. Wine from strain AWRI2940 was notably higher in ‘rose aroma’, as well as in ‘yellow colour’ and ‘sourness’, while lower in ‘cooked vegetable/potato’ and ‘sweetness’. Strains AWRI2936, AWRI2965, and AWRI4124 were rated with intermediate values for ‘rose aroma’  was used to investigate the relationships between wine composition and sensory attributes for both time points at which the wines were evaluated . Chemica3H, methionol), and the compounds 2-PEA, ethyl butanoate, 2- and 3-methylbutyl acetate, 2-methylpropanol, 3-methylbutanol were significant contributors to the sensory differences and were heavily loaded on Factor 1.Both the 3-month and 15-month PLS-R models indicated three optimum factors, explaining 50% and 54% of the variance of the sensory data, respectively. 2 calibration 0.69 and R2 validation 0.51), ‘floral aroma’ , ‘cooked vegetable/potato aroma’ , ‘sweetness’ , ‘viscosity’ , and ‘stone fruit flavour’  were relatively well modelled but not so well predicted, indicated by the low R2 validation. TOL and TOL-SO3H were significantly associated with ‘yellow colour’. No compounds were significant for ‘floral aroma’ but two monoterpenes, cis-rose oxide and α-terpineol, and several esters had moderately sized positive regression coefficients  for this attribute, while 2-PE and 2-PEA were only weakly positively associated. ‘Cooked vegetable/potato aroma’ was associated with several sulfur compounds, notably H2S and MeSH. ‘Bitterness’ was most strongly related to volatile acidity and 2-phenylacetaldehyde, with TyrOH and TOL-SO3H weakly associated. TOL-SO3H and volatile acidity, in addition to 2-PE and 2-PEA, were significantly negatively associated with ‘sweetness’.Of the sensory attributes at the 3-month time-point, ‘yellow colour’ , ‘stone fruit aroma’ , ‘rose aroma’ , ‘cooked vegetable/potato aroma’ , ‘sweetness’ , ‘stone fruit flavour’ , ‘rose flavour’ , and ‘sourness’  were relatively well modelled, while ‘banana confection aroma’, ‘citrus flavour’, ‘pungent aroma’, and ‘bitterness’ were not modelled well. Similar to the model at three months, TOL and TOL-SO3H were again significantly associated with ‘yellow colour’, together with several other compounds. Of the many compounds associated with ‘rose aroma and flavour’, 2-PE and 2-PEA had the largest positive regression coefficients, while the volatiles 2-phenylacetaldehyde, methionol, hexyl acetate, 2- and 3-methylbutyl acetate, ethyl butanoate, 2-methylpropyl acetate, ethyl 2-methyl propanoate, H2S, and non-volatiles TOL and TOL-SO3H were also associated with these attributes. There was strong evidence for an association between ‘cooked vegetable/potato aroma’ and the compounds methional (regression coefficient 0.09) and MeSH (regression coefficient 0.07); although the association with H2S was weak, it had a relatively high regression coefficient of 0.04. There was strong evidence for a negative association between the compounds TOL and TOL-SO3H and ‘sweetness’, and a positive association with ‘sourness’. Although ‘bitterness’ was not well modelled, TOL and TOL-SO3H were positively associated with this attribute, with relatively high regression coefficients.In the 15-month PLS model, similar links between compounds and sensory attributes emerged, but this model was generally stronger and predicted most attributes well. From the three-factor model, ‘yellow colour’  is consistent with that reported in a range of commercial white wines of different ages [2 and TOL was between 13 and 132 in wines with low to moderate TOL concentrations favouring the formation of TOL-SO3H. The high conversion yields are therefore not unexpected. In the wines made with the highest TOL producer, AWRI2940, this molar ratio was as low as 1.8, explaining the lower conversion yield. The TOL concentration found in Chardonnay wines fermented with the low and moderate TOL-producing strains was in the range of those reported in the literature for commercial white wines, typically below 5 mg L−1 [2 due to this reaction would be less than 2 mg L−1. A survey of a large number of commercial bottled Australian white wines [−1 of free SO2 in the most recent vintages assessed, a concentration similar to that found in our Chardonnay wines at bottling (39 mg L−1). These data suggest that the conversion of TOL into TOL-SO3H makes only a limited contribution to the losses of SO2 observed during wine development, which are primarily driven by reaction with dissolved O2 in wines after bottling and by sulfonation of other species [3H concentration and SO2 loss in wine observed here.Our results demonstrate that the time required to reach equilibrium between TOL and its sulfonated adduct, typically between 3 and 6 months, is independent of the initial amount of TOL. Longer storage times have little or no effect on the yield of TOL-SOent ages . The mol5 mg L−1 ,34,35. Ate wines  found an species ,36. This3H. This high accumulation allowed us to assess the possible effects of TOL and TOL-SO3H on white wine sensory properties. After 15 months in-bottle, wines made with AWRI2940 were rated higher in ‘sourness’ and lower in ‘sweetness’, and tended to be more bitter for some assessors even though these wines had slightly elevated fructose and glycerol concentrations  relative to the control. The PLS-R results suggest that both TOL and TOL-SO3H may impart a degree of ‘bitterness’ to these wines while decreasing ‘sweetness’ and increasing ‘sourness’ ratings, probably through well-established taste–taste interactions [Wines made with AWRI2940 accumulated exceptionally high concentrations of both TOL and TOL-SOractions , especia3H in a wine matrix have been published, but Van Gemert [−1) eliciting bitter taste was reported in water [−1) [−1) have been reported to impart bitterness in wine [−1 of TyrOH to white wine and observed no influence on sensory properties, and only when unrealistically high concentrations were added (500 mg L−1) did TyrOH seem to depress the overall ‘flavour quality’ of wine. The fact that our wines made with AWRI2965, with a concentration of TyrOH exceeding 120 mg L−1, were not rated differently to the control wine in palate attributes such as ‘astringency’, ‘bitterness’ or ‘sourness’, suggest a limited effect of this higher alcohol on white wine in-mouth sensory properties.To our knowledge, no sensory recognition or detection thresholds for TOL-SOn Gemert  lists a n Gemert ,14,30, cn Gemert . Consequn Gemert . Differein water , lower ater [−1) . Even th in wine , to the  in wine  added 50Over time in-bottle, the sensory differences among the wines produced by each yeast became larger. Although the magnitude of the strain effect on ‘floral/rose aroma’ was similar across the two time points , an aromAnother aspect related to the effect of aging is the formation of aldehydes from oxidation of the analogous higher alcohols. These aldehydes can have a much lower sensory threshold than their corresponding higher alcohols; for example, the sensory threshold for methional is about 1000-times lower than that of methionol ,42. TherARO8 in a wine strain, encoding for the aromatic transaminase Aro8p, decreased methionol formation after fermentation of a synthetic grape must [ARO8 mutants indicates that by blocking the aromatic Ehrlich pathway, the catabolism of methionine might also be impaired.The production of methionol and other negative VSCs has been shown to be affected by the branch of the Ehrlich pathway responsible for the production of aromatic higher alcohols . Recentlape must . The decARO9 and ARO10 genes are highly induced by aromatic amino acids [ARO9-promoter-BFP reporter gene  it can be described as contributing ‘blackcurrant’ and ‘red fruit’ aromas, and it is considered to enhance the bouquet of some wine styles [The idea that there is competition for methionine between the Erhlich and demethiolation pathways is supported by the VSC profile observed in the pilot-scale wines made with the three homozygous Tyr1p mutants, particularly with the most active strain AWRI2940. Again, AWRI2965, which harbours a mutation (Aro4p) earlier in the amino acid biosynthetic pathway, behaved somewhat differently than the Tyr1p variants. Higher concentrations of methionol were not observed in the wines produced using AWRI2965 but decreased concentrations of other undesirable VSCs such as MeSH, MeSAc or methional were observed. Interestingly, AWRI2965 also produced slightly higher concentrations of DMS in these conditions. Even though DMS is also associated with reductive off-odours and ‘vegetal’ aromas, at low concentrations  culture collection . These v−1 of methionine. SGM was filtered through 0.22 µm Stericup filters (Millipore). Yeast starter cultures were prepared by growing cells aerobically in YPD medium for 24 h to stationary phase at 22 °C. Then, 1 × 106 cells mL−1 were inoculated into 50% diluted SGM medium and grown for another 48 h at 22 °C. The acclimatised cells were inoculated into 100 mL of SGM at a density of 1 × 106 cells mL−1. Fermentations were conducted at 17 °C in 100 mL glass bottles (Schott Duran), fitted with stir bars and stirred at 200 rpm using a magnetic stirrer. The lids of the bottles were fitted with selective H2S detector tubes  to measure the release of H2S during fermentation. Fermentation progress was followed by CO2 weight loss, measured every 24 h. After fermentation, the wines were cold settled at 4 °C for 5 days and sampled for different volatile and non-volatile compound analyses.Laboratory-scale fermentations were performed in triplicate in a synthetic grape medium (SGM) , with a −1, and pH 3.29. A concentration of 25 mg L−1 of SO2 was added to the grape must at the crusher. Yeast strains were grown for 48 h in filter-sterilised neutral grape concentrate , which had been previously diluted to ~6 °Baume and pH adjusted to 3.5. Cells were inoculated at a density of approximately 2 × 106 cells mL−1 in 19 L of the Chardonnay juice, and fermentation was conducted at 15 °C in 20 L stainless steel kegs in triplicate. When °Baumé was below 3, wines were moved to 20 °C. Irrespective of the starter culture used, wines got stuck around 1 °Baumé (day 16–18 of fermentation). Ferments were then rescued at day 26 by the addition of the commercial yeast Lalvin EC 1118 . Once alcoholic fermentation had finished (day 31), wines were sulfured with 80 mg L−1 of SO2, and cold-stabilised for approximately 2 months at 0 °C. Before bottling, SO2 concentration was adjusted to between 35–40 mg L−1 of free SO2. Screw-cap sealed bottled wines (375 mL) were stored in the dark at a constant temperature of 15 °C.The pilot-scale winemaking trial with Chardonnay juice was performed by the Wine Innovation Cluster (WIC) winemaking services, according to a standardised white winemaking protocol. Hand-harvested Chardonnay grapes from the McLaren Vale region  were used. The basic chemical parameters of the Chardonnay juice were: 12.7 °Baumé, yeast assimilable nitrogen 217 mg LTargeted analyses of fermentation-derived compounds  were performed by Metabolomics Australia (Adelaide) by GC-MS using a stable isotope dilution assay  at the ecis-rose oxide, α-terpineol, nerol, geraniol) and C13-norisoprenoids (β-damascenone and β-ionone) was performed at 3 months post-bottling by GC-MS on an Agilent 6890 gas chromatograph equipped with a Gerstel MPS2 autosampler and coupled to an Agilent 5973N mass selective detector. Sample preparation was as follows: 10 mL of wine was transferred into a 20 mL crimp-cap, headspace-SPME vial (Grace Davison) with 3 g of NaCl followed by 50 µL of a combined d4-β-damascenone, d3-α-ionone and d3-β-ionone internal standard solution. Instrument control was performed with Agilent G1701EA Revision E.02.02 ChemStation software. The gas chromatograph was fitted with an Agilent DB-5ms 30 m × 0.25 mm × 0.5 um. Helium (Ultra High Purity) was used as the carrier gas with linear velocity 46 cm/s, flow rate 1.6 mL/min in constant flow mode. The oven temperature was started at 40 °C, held at this temperature for 2 min, then increased to 190 °C at 8 °C/min and held at this temperature for 5.25 min. The vial and its contents were heated to 60 °C for 10 min in the heater/agitator with the agitator on for 5 s and off for 2 s at 500 r.p.m. A Supelco grey 2 cm SPME fibre was exposed to the sample during this heating time through the septum. The fibre was then injected into a split/splitless inlet in splitless mode. The analytes were desorbed into a Supelco 0.75 mm ID sleeveless SPME liner for 10 min, which was held at 200 °C. The purge flow to the split vent was 50 mL/min at 2.1 min with the septum purge flow turned off. The mass spectrometer quadrupole temperature was set at 150 °C, the source was set at 230 °C and the transfer line was held at 250 °C. EMV Mode was set to Gain Factor = 1.00 and spectra were recorded in SIM mode.Analysis of monoterpenoids , as described previously [2S and MeSH, respectively. Ethylmethyl sulfide and propyl thioacetate were used as internal standards. Analytes were identified by comparison of their retention times with those of the corresponding pure reference compounds.The VSCs Heviously . ReferenO-hydroxylamine hydrochloride (Sigma-Aldrich). Reference standards for these compounds of the highest purity were purchased from Sigma-Aldrich. Isotopically labelled analogues for furfural, methionol, methional, benzaldehyde, and 2-phenylacetaldehyde were used as internal standards for accurate quantification of these compounds. For the quantitation of 2-methylpropanal and 3-methylbutanal, d5-benzaldehyde was used as an internal standard. Similarly, d4-furfural was used for the determination of 5-methylfurfural. With the exception of d4-furfural , the synthesis of the other isotopically labelled standards was carried out in-house as described in [Analysis of the sulfur-containing compounds methionol and methional, as well as 2-methylpropanal, 3-methylbutanal, furfural, 5-methylfurfural, benzaldehyde, and 2-phenylacetadehyde was performed by GC-MS/MS, as described in . Aldehydribed in .VSCs and aldehydes were analysed 3 and 15 months post-bottling.The concentrations of sugars, ethanol, glycerol, and organic acids  were measured by HPLC using a Bio-Rad HPX-87H column, as described previously . Referen3H were analysed on an Agilent 1200SL HPLC using a Phenomenex Kinetex PFP column  at different time points . The injection volume was 5 µL. The column was eluted at 45 °C with a gradient of 0.1% formic acid in Milli-Q water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.4 mL min−1. The gradient was as follows: an initial isocratic hold (0% B) for 8 min, then gradient to 5% B over 32 min, gradient to 25% B over 9 min, then gradient to 80% B over 3 min, held isocratically at 80% B for 3 min, and dropped to 0% B and held for another 15 min. Absorbance at 280 nm was monitored with an Agilent 1260 Series G7117C DAD, while fluorescence was monitored at excitation and emission wavelengths of 280 and 350 nm, respectively, with an Agilent 1260 Series G7121B FLD. Quantification of TOL and TyrOH was performed using the absorbance detector, while TOL-SO3H was quantified using the fluorescence detector. Reference standards for TyrOH and TOL were obtained from Sigma-Aldrich. TOL-SO3H was synthesised as previously described by Arapitsas, Guella and Mattivi [2O) with stirring and reacted at room temperature for 48 h. The reaction product was dried under a vacuum (30 °C) and dissolved in H2O. The product was purified using preparative HPLC with a Dionex UltiMate 3000 system, a C18 Synergi Hydro RP column , and solvent system of 100% H2O (A) and 100% acetonitrile (B). Gradient: 0–10 min 0% B, 10–25 min 50% B, 25–35 min 100% B, 8 mL/min flow rate. The structure of TOL-SO3H was confirmed using HRMS and NMR  with samples in D2O at 300 K. Results were processed using Topspin software. M-H mass (m/z): 240.0325; Chemical shifts for 1H-NMR  and 13C-NMR (D2O) concur with those previously reported [TyrOH, TOL, and TOL-SO Mattivi  with modreported .Quantitative descriptive analysis (QDA) sensory studies  were conp values were determined by a two-tailed Student’s t test. For the sensory data, ANOVA was carried out using Minitab 19. The effects of the yeast strain treatment, judge, judge by strain, ferment replicate nested into strain, judge by ferment replicate nested into strain, presentation replicate nested into strain, and ferment replicate were assessed, treating judge as a random effect. Following ANOVA, a protected HSD value was calculated using the mean square term of the judge × strain interaction at a 95% confidence level for attributes with a significant (p < 0.05) treatment effect. To explore the relationship between wine chemical composition and sensory attributes, PLS-R was conducted for each wine replicate, as described in [p < 0.10) signalled a treatment effect or a high F-ratio was found indicating potential treatment effects which may have been overshadowed by judge, fermentation, or presentation replicate variation.Minitab 19  was used for statistical analysis of the compositional data which were analysed by one-way analysis of variance (ANOVA). Multiple comparisons of the analyte concentration with respect to treatment were undertaken using Tukey’s honestly significant difference (HSD) test , and ribed in  with somThis study confirmed that the higher alcohol overproduction phenotype of five variants derived from the commercial wine strain AWRI796 is maintained in pilot-scale white winemaking conditions. This overproduction was associated with meaningful changes in wine sensory profiles, especially after some period of bottle storage. The effect of these strains on wine chemical composition was not just limited to the overproduction of 2-PE but also to an increase in the concentration of the higher alcohols TyrOH and/or TOL and to the formation of VSCs. Associations between these compounds and ‘sweet’, ‘sour’ and ‘bitter’ tastes, and ‘cooked vegetable/potato aroma’, were identified. These results highlight the intricate connections between the metabolism of aromatic amino acids and the sulfur-containing amino acid methionine during fermentation, ultimately influencing wine flavour.The various yeast strains isolated in this study provide novel tools for winemakers to adjust and preserve wine style. In particular, AWRI2965 has excellent potential as a white wine winemaking yeast, imparting accentuated and lasting rose/floral aromas to wines.3H, needs to be elucidated along with the physico-chemical conditions such as pH, temperature, storage time, and SO2 concentration, which might influence the equilibrium between these compounds in the finished wine.More research will be needed to understand the compositional drivers of bitterness. In particular, the role of higher alcohols derived from the metabolism of aromatic amino acids TyrOH, TOL, and its sulfonated derivative TOL-SO


Class III peroxidases constitute a plant-specific multigene family, where 73 genes have been identified in Arabidopsis thaliana. These genes are members of the reactive oxygen species (ROS) regulatory network in the whole plant, but more importantly, at the root level. In response to abiotic stresses such as cold, heat, and salinity, their expression is significantly modified. To learn more about their transcriptional regulation, an integrative phenotypic, genomic, and transcriptomic study was executed on the roots of A. thaliana Pyrenean populations. Initially, the root phenotyping highlighted 3 Pyrenean populations to be tolerant to cold (Eaux), heat (Herr), and salt (Grip) stresses. Then, the RNA-seq analyses on these three populations, in addition to Col-0, displayed variations in CIII Prxs expression under stressful treatments and between different genotypes. Consequently, several CIII Prxs were particularly upregulated in the tolerant populations, suggesting novel and specific roles of these genes in plant tolerance against abiotic stresses. The CI duce ROS .Therefore, they are involved in a myriad of physiological processes throughout the plant’s life cycle because of their genetic and catalytic diversities . They coPrx62 was found to be implicated in salt tolerance during germination [CIII Prxs enhanced cold  [Prx08) [Moreover, several CIII Prxs were characterized for their significant roles in providing tolerance to plants against abiotic stresses. For instance, mination . Additio [Prx08) . Indeed,A. thaliana [The presence of 73 members in thaliana  suggeststhaliana . The attthaliana , and thethaliana .CIII Prxs. Notably, these transcriptional regulations are not linear circuits but are rather integrated networks involving multiple actors, where thousands of genes are differentially regulated under abiotic stresses [With the increasing manifestations of climate change, plants are facing more frequent and severe abiotic stresses that drastically affect their growth and development ,12,13. Pstresses .A. thaliana has been adopted as a model species [A. thaliana Pyrenean ecotypes to analyze the transcriptional regulation of CIII Prxs in response to multiple abiotic stresses and in different populations. By definition, natural intraspecific variation is the inherent genetic diversity among individual organisms making up a population within a species. It originates from the accumulation and natural selection of spontaneous mutations that improve the fitness of their hosts to their direct environment [ species  because  species . This stironment . It was ironment ,23,24,25A. thaliana populations, distributed along an altitudinal gradient in the French Pyrenees, not covered yet in the 1001 Genomes Project [CIII Prxs regulation under multiple abiotic stresses. The objective of this study is to provide a detailed view of the roles of CIII Prxs in response to abiotic stresses in A. thaliana Pyrenean populations.Therefore, this study implemented the natural intraspecific variation of 30  Project , in addiThe root development of the populations was studied under control conditions “ctrl” (22 °C) and under three stresses: cold stress “C” (16 °C), heat stress “H” (28 °C), and salt stress “S” (22 °C + 50 mM NaCl). Accordingly, three stress-tolerant populations were identified based on their root phenotypes under each treatment. Then, the seed germination of these selected populations was tested to mark the initiation of their root development. Afterward, transcriptional analyses were performed on Col-0 and the selected stress-tolerant populations to highlight the differential regulation of CIII Prxs in response to abiotic constraints.A. thaliana populations, and on the other hand, the differential impacts of temperature and salt stress on this trait among the studied populations.The primary root growth of the multiple populations under different growth conditions was described by the root growth rates “RGRs” (mm/day) and the primary root lengths “PRLs” (mm). The aim was to highlight, on the one hand, the natural plasticity of the root development in the different p-values less than 0.0001  of the populations under the four designed growth conditions were summarized using principal component analysis (PCA). The principal component 1 (PC1) explained 54% of the phenotypic variables, while PC2 explained 20%. The populations were differentially distributed along the PCA plot, and they mostly clustered apart from Col-0 and Sha, which highlighted their plasticity in response to abiotic stresses d. PC1 isThe development of the populations’ primary roots was tracked over 8 to 14 days, and it was contrasted in a genotype-dependent as well as treatment-dependent manner . For insAt 16 °C, Eaux, Belc, and Roch developed the longest roots  at the fastest rates , whereas Mari, Biel, and Bedo grew the shortest ones  most slowly . Interestingly, certain populations, such as Hosp and Mere, had moderate PRLs (10.14 and 10.34 mm) but with fast growth rates (0.88 and 0.86 mm/day). This phenomenon was due to the slow rates of seed germination of these populations, which delayed the root development initiation .To further highlight the impacts of the applied stress on the populations, the PRL of each population under each treatment was compared to that at 22 °C (ctrl). Under cold stress, the PRLs of all the populations decreased, yet the magnitudes of these reductions were differential according to the genotype. For instance, the PRLs of Eaux, Hern, and Lave were the least reduced at 16 °C compared to Arag, Argu, and Bedo . AccordiAt 28 °C, the most elongated roots and fastest growth rates were observed in Urdo, Jaco, and Herr ; however, Biel, Mari, and Hern had the shortest roots  with slowest RGRs . These observations revealed that the temperature differentially affected the root growth of the studied populations and that its effects were influenced by the different genotypes (populations) .Under heat stress, several populations developed longer roots compared with the ctrl, such as Herr, Urdo, and Jaco, while others developed shorter ones, such as Col-0, Roch, and Hosp . Herr waMoreover, Hosp, Prad, and Guch developed the longest roots under salt stress , with higher growth rates for Hosp and Guch (1.46 and 1.40 mm/day) but a lower one for Prad (1.01 mm/day). Yet, the roots of Bier, Mere, and Mari were reduced because of salinity  . AltogetFurthermore, the salt treatment globally decreased the PRLs and RGRs; however, these reductions were variable, depending on the population. To exemplify, the PRLs were moderately diminished in populations such as Savi, Prad, and Grip compared with other severely reduced populations such as Gedr, Bier, and Mere . CorrespTo sum up, the root phenotyping showed that the populations had varying root growth under control conditions which could be related to their genetic architecture. Additionally, their roots were generally reduced because of the applied treatments, except under heat for some populations. Most importantly, they unevenly responded to the applied stresses, where the root development of sensitive populations was more reduced compared with other tolerant ones. Accordingly, the root phenotyping allowed identifying some populations that had fairly advanced root growth under cold (Eaux), heat (Herr), and salt (Grip) stresses compared with Col-0. Then, the seed germination of the selected populations was tested to benchmark the beginning of their root development, aiming to know if they developed longer roots because they formerly germinated earlier or because they were truthfully tolerant to the stresses.Germination tests were performed on the cold-, salt-, and heat-tolerant populations  in addition to Col-0 under different combinations of treatments. These experiments highlighted the existing natural plasticity of this trait among the selected candidates and featured how cold, heat, and salt altered it. In addition, it was essential to assess the germination rates of the populations to normalize the effects of any significant variations that may advance or delay the initiation of root growth. For this aim, the testa rupture (TR) and the endosperm rupture (ER) were tracked over a time course . The gerIn Col-0, 100% of the seeds successfully germinated under the four conditions; however, the speed of their germination increased with the temperature but decreased with the salt a,b. To iThese results suggested that the germination of Col-0 was fastest at 28 °C, but it decelerated at 16 °C and under salt stress. Additionally, the low temperature slowed the germination of Col-0 more than the salinity. Moreover, the cold stress reduced the TR more than the ER; the heat stress enhanced the TR and ER almost equally, whereas the salt stress decelerated the ER more than the TR.The seeds of Eaux, the cold-tolerant population, fully germinated under the ctrl and cold conditions. At 22 °C, the TR and ER t50s recorded 43.46 and 55.92 h. They both increased under the cold treatment by 45% and 56%. Furthermore, the germination of Eaux was constantly slower than that of Col-0 under the two tested conditions. To illustrate, the TR t50s of Eaux were prolonged by 20.47 and 39.9 h compared with those of Col-0 at 22 °C and 16 °C, and its ER t50s were extended by 26.07 and 57.61 h under ctrl and cold conditions compared with Col-0 c,d.Herr, the heat-tolerant population, entirely germinated at 22 °C and 28 °C. The TR and ER t50s recorded 51.67 and 65.03 h at the control conditions. The heat treatment (H) did not significantly decrease the TR t50 (6%), but it diminished the ER t50 by 16%. In addition, the germination of Herr was always lagging that of Col-0. For instance, the TR t50s in Herr were greater than those in Col-0 by 28.68 and 25.8 h at 22 °C and 28 °C, respectively, and its ER t50s were longer by 35.18 and 24.65 h under ctrl and heat conditions compared with Col-0 c,d.Grip, the salt-tolerant population, completely germinated under the ctrl and salt conditions, although salinity delayed its germination. To illustrate, its TR and ER t50s were 41.13 and 35.29 h at 22 °C. Upon salt application, they increased by 20% and 65%, respectively. In reference to Col-0, Grip had slower germination under both conditions. To illustrate, the TR t50s were prolonged by 18.14 h and 26.16 h, and the ER t50s by 5.44 h and 28.24 h, respectively, under the control and salt stress c,d.To sum up, the tests showed that each of the selected populations  had slower seed germination compared with Col-0 under both the control and the corresponding stress condition. However, the slowness in their germination was extended because of the different stresses, causing a subsequent delay in their root development initiation. However, despite these delays, the roots of Eaux, Grip, and Herr were less reduced compared with Col-0 under cold, salt, and heat treatments, respectively. Hence, their choice as convenient candidates for cold, salt, and heat tolerance was validated.CIII Prxs were extracted  under control conditions (22 °C) and under the corresponding stress for each, i.e., cold for Eaux, heat for Herr, and salt for Grip. The overall results were summarized in gene-level expression tracks (GEs) holding information about the expression values (TPMs) for each gene in the multiple populations grown under different conditions . The expxtracted .CIII Prxs under control conditions (22 °C) varied within and among the studied populations. For instance, a subset of five CIII Prxs was highly expressed in all populations , with an average of 95  were significantly upregulated, while 15 genes were downregulated because of the cold treatment compared with the ctrl. However, nine members of this multigene family were upregulated in Eaux under cold, where only Prx71 was mutual with Col-0. In addition, 10 genes were downregulated in Eaux under low temperature, where Prx38, Prx52, Prx53, Prx54, and Prx65 were exclusively suppressed in this population. Comparing genotypes, 17 upregulated CIII Prxs, and 14 downregulated ones were present in Eaux versus Col-0  was specifically assayed using guaiacol/H2O2  in Col-0These results showed that CIII Prxs had basal enzymatic activity in all studied populations under control conditions, proving that these proteins performed vital processes during cellular homeostasis. Their activity was importantly enhanced by heat in Col-0, which displayed their role in responding to temperature changes. Additionally, the low-temperature treatment in Eaux induced their activity, suggesting that they could contribute to cold tolerance. However, it did not increase in Herr, the heat-tolerant population, when exposed to elevated temperatures.CIII Prxs expression and their peroxidase activity was noticed. To illustrate, a subset of 7 CIII Prxs was upregulated in Eaux, whereas only two of these genes were upregulated in Col-0 under the cold treatment. In addition, Eaux had more upregulated CIII Prxs at 16 °C when compared with Col-0 based on their genotypic difference. Coherently, this transcriptional pattern was reflected in the peroxidase activity, which was enhanced in Eaux under cold stress. Similarly, under hot conditions, 11 CIII Prxs were upregulated in Col-0, but only the expression of Prx65 was enhanced in Herr. Moreover, the genotype-based DE analyses detected 12 upregulated genes in Col-0 versus Herr at 28 °C. Consistently, the peroxidase activity was triggered by elevated temperatures in Col-0 but not in Herr.A correlation between the A. thaliana, where numerous genomic maps of climate adaptations were established for populations at macro and microgeographic scales [On an evolutionary scale, plants adapted to their local environments by selecting genes that enhanced their fitness  and expac scales .A. thaliana, so they could serve as new tools to understand the genetic variation and the plant adaptations to abiotic stresses between the Iberian Peninsula and the rest of Europe. The particularity of this sampling was derived from their close geographical distribution but in highly contrasted environments due to the mountainous nature of their collection sites. To illustrate, these populations spanned an altitudinal gradient, and hence they were naturally exposed to variable climatic conditions such as annual minimum, maximum, and mean temperatures (°C), in addition to annual precipitation (mm) and total annual UV radiations (kWh/m2). Such variations would certainly trigger the evolutionary divergence among these populations.The Pyrenean populations in this study filled a gap in the geographic distribution of In the Pyrenees Mountains, the local environmental conditions are highly contrasted, where temperature, precipitation, and UV radiation patterns are correlated with the altitude. In a previous study, the genetic structure of the populations was established, by which they were segregated into three clusters, and specific genetic lineages were detected among them. This genetic diversity was translated into contrasted phenotypes of these populations when exposed to suboptimal temperatures .In this study, the populations’ roots phenotypes were also contrasted between different abiotic stresses. The root lengths and growth rates were used as indices to estimate the root development in the populations. In fact, roots play fundamental roles in the plant by foraging underground resources such as minerals and water. They are also known for their enormous plasticity by which they can respond to extrinsic cues via modulating their growth . TherefoTo sum up, differential root lengths and growth rates were observed between different treatments and various populations. The multivariate analyses of the root phenotypic data allowed the classification of the populations into cold-, heat-, and salt-tolerant or sensitive. For instance, Arag and Bedo were cold-sensitive populations since they developed tiny roots at 16 °C, but Eaux and Lave were cold-tolerant populations having relatively longer and faster-growing roots under cold conditions. These cold-sensitive and tolerant populations were formerly characterized to accumulate low and high anthocyanin content, i.e., stress indicator, in their rosettes under cold stress .Moreover, the seed germination of Col-0 and selected stress-tolerant populations  was studied under control and stressful conditions, which determined their germination timing and hence the beginning of their root development. This experiment enabled distinguishing whether the stress-tolerant populations developed longer roots because of early germination and growth initiation or because of their potential to develop properly under stress.In fact, seed germination is a crucial event that marks the beginning of the plant’s life cycle and ensures its survival . It is rThe seeds of Col-0, Eaux, Grip, and Herr fully germinated under control and under stressful conditions, but their germination speed varied between genotypes and treatments. For instance, Col-0 had the fastest germination rate compared with the selected Pyrenean populations under all tested conditions. However, the selected populations developed longer roots under stressful conditions compared with Col-0 despite the delay in their growth initiation caused by the delay in their seed germination. This observation validated that these populations were stress tolerant regarding their root development since they developed longer roots faster compared with Col-0. Indeed, these observations highlighted the phenotypic plasticity among the Pyrenean populations, which could be related to their genetic diversity.RNA-seq was adopted as a high-throughput technique to study the whole transcriptome of an organism at once . TranscrCIII Prxs in three stress-tolerant Pyrenean populations, in addition to Col-0, under control and stressful conditions. In fact, CIII Prxs is a larger gene family including a plurality of members compared with ascorbate peroxidase  and glutathione peroxidase  [CIII Prxs were previously characterized as extensively involved in many physiological processes and play key roles during the plant’s response to environmental constraints [This study focused on the expression of the amilies) . In addistraints , during straints .CI Prxs, only a few genes were found significantly differentially expressed in the Pyrenean populations. To exemplify, the treatment-based DE analyses showed that GPx02 and GPxO6 were upregulated in Eaux under cold stress compared with the optimal condition, and APx01 was enhanced in Grip under salinity. Yet, none were triggered in Herr when exposed to heat. Additionally, the genotype-based analyses highlighted other CI Prxs with higher expression in the Pyrenean populations compared with Col-0, such as APx01 and GPx07 in Eaux at low temperature, APx01 and APx04 in Herr at high temperature, and APx01 in Grip at saline conditions. Despite such significant changes in the expression of APxs and GPxs between genotypes and in response to abiotic stresses, the variations detected in CIII Prxs were more multitudinous.As for CIII prxs transcriptomic data under control conditions vividly demonstrated the natural variation in their expression between the Pyrenean populations, which could be attributed to their underlying genetic diversity. Moreover, the treatment-based differential expression (DE) analyses showed that the expression of many CIII Prxs was altered in response to the applied stress in each population. Additionally, the genotype-based DE analyses displayed that their regulation was variable between the stress-sensitive Col-0 and the stress-tolerant Pyrenean populations under each condition. Consequently, their contrasted expression between treatments and genotypes allowed the characterization and specification of novel roles of these genes in providing tolerance to plants.Initially, CIII Prxs in Col-0 and Eaux, with Prx71 mutually upregulated in both populations. These results suggested novel roles in cold tolerance of the Eaux-exclusive eight upregulated genes . Furthermore, another subset of 17 upregulated CIII Prxs in Eaux compared with Col-0 was identified by the genotype-based DE analyses, suggesting putative roles of 9 additional genes to be involved in cold tolerance   analyses highlighted subsets of two and nine upregulated d Prx70) a.CIII Prxs in Col-0 and Herr, with Prx65 mutually upregulated in both populations, signifying that these genes were involved in heat tolerance . Additionally, the genotype-based DE analyses, which highlighted a subset of six CIII Prxs to be upregulated in the heat-tolerant population “Herr”, suggested additional putative roles of these genes in response to elevated temperatures   a.CIII Prxs in Col-0 and Grip, with Prx49 mutually upregulated in both populations, proposing new functions of the Grip-exclusive upregulated genes in the response against salty conditions (Prx15 and Prx62). Furthermore, the genotype-dependent DE analyses, which exposed a subset of 11 upregulated CIII Prxs in the salt-tolerant population “Grip” compared with Col-0, suggested additional putative roles of these genes in response to salinity   a.CIII Prxs. For instance, some CIII Prxs were significantly upregulated in the cold-tolerant population Eaux compared with Col-0 under low temperature  or in Eaux at 16 °C compared with 22 °C (Prx55 and Prx57), suggesting that they were particularly involved in cold tolerance. These genes were previously characterized for other roles such as Prx16, which was involved in seed germination [Compiling these results allowed a functional specification of temperature- and salt-related mination , Prx27 imination , Prx47 imination , Prx66 imination . Additiomination  and Prx5mination  , or in either Col-0 or Herr at 28 °C compared with 22 °C  were associated to the response against heat stress. Formerly, Prx61 and Prx67 were not associated with any functions in any context. However, Prx17 was a direct target of the AGAMOUS-LIKE15 transcription factor and contributed to lignification [However, genes that were upregulated in the heat-tolerant population Herr compared with Col-0 under high temperature , in Grip at salty conditions compared with ctrl conditions (Prx15 and Prx62), or in Col-0 (Prx49 and Prx52) had specific roles in responding against salinity. The corresponding proteins were formerly known for playing distinct roles in plants, except for Prx14. For instance, Prx40 was found essential to proper anther and pollen development [Nevertheless, other elopment . As for elopment , while Pelopment . Moreoveelopment  and in lelopment , respectPrx01, Prx37, and Prx60 had higher expression under both cold and hot temperatures, and they hence played roles in responding to temperature variations. Formerly, Prx01 was identified as an actor in pathogenesis, cold tolerance, and extension regulation during root hair growth [Botrytis cinerea [In addition, some genes were upregulated under different treatments in the Pyrenean populations suggesting that they were involved in numerous stress-induced physiological responses. To exemplify, r growth , Prx37 i cinerea , and Prx cinerea  and the  cinerea   gene regulating root hair development [Furthermore, elopment . Prx10 welopment , while Pelopment   was triggered by all applied stresses, i.e., cold, heat, and salinity. These genes were previously identified in other contexts, such as Prx08, which was involved in regulating epidermal differentiation in Arabidopsis roots [Finally, a subset of three is roots . Moreoveis roots , while Pis roots   and Shahdara . Plant individuals of these populations were previously gathered from different locations in the French Pyrenees, the mountainous physical barrier separating the Iberian Peninsula from France. Their names, climatic data, and taxonomic relevance to A. thaliana were previously reported [Thirty new-found reported . To mini2·s−1).The primary roots lengths of the 32 studied populations were measured over a timeline under four growth conditions. Plants were grown in vitro in vertical positions inside 12 × 12 square Petri dishes at four contrasted conditions on half Murashige and Skoog basal medium (½ MS) with sucrose and agar  and with the addition of 50 mM NaCl  when salt stress was applied. The seeds were sterilized with a bleach solution , and 0.1% Triton), followed by three successive washes with autoclaved water. For each biological replicate, 16 to 18 seeds per population were sown along two lanes with even spacing; each lane contained 8 seeds from 2 populations to maximize homogeneity and randomness. Afterward, a cold stratification treatment was performed at 4 °C for 24 h in the dark to synchronize and promote germination before transfer to long day (16 h light/8 h dark) growth chambers (light intensity = 150 µmol/mThe plants were grown at three different temperatures designating a control “Ctrl” (22 °C), cold stress “C” (16 °C), and heat stress “H” (28 °C). They were grown with 50 mM NaCl at 22 °C . The plants were scanned on a flat-bed scanner  at 1200 dpi over a timeline adapted for each temperature. The scanning time points were 0, 3, 6, 8, 10, and 14 days at 16 °C; 0, 2, 3, 4, 6, 8, and 10 days at 22 °C; and 0, 2, 3, 4, 6, and 8 days at 28 °C.The primary roots were measured using the NeuronJ plug-in in ImageJ. The experiments were carried out in 3 to 12 replicates. Most data analyses were performed using the R software (Version 1.2.1335). First, the root growth rates RGRs (mm/day) were computed as the slopes of the linear regression lines of the primary root lengths (PRLs) measurements (mm) as a function of time (days). Second, the arithmetic means and the standard errors of means . Then, the differences among the PRLs recorded on the last day of growth were measured in percentage to evaluate their variation in each population and under each treatment in reference to the control (22 °C). Additionally, the variations in the PRLs and the RGRs due to the different treatments in the populations were statistically tested using the analysis of variance, two-way ANOVA, from the “multcomp” R package .Germination tests were performed on seeds of three selected cold-, heat-, and salt-tolerant populations , in addition to Col-0, to estimate the effects of the corresponding treatments and different genotypes on seed germination. The germinating seeds were quantified up to 144 h. The three stages of germination were no rupture of the seed envelope (NR), rupture of the external testa envelope (TR), and rupture of the internal endosperm envelope (ER) marked by the embryo’s radicle protrusion. The statistical analyses were performed using the “germinationmetrics” R package .2·s−1). Col-0 germination was tested under all conditions, whereas each of the three selected Pyrenean populations was tested under the control (22 °C) and the stress condition to which it was tolerant. The quantification was performed using a binocular loupe . The experiments were conducted in triplicates.The germination tests were performed in vitro inside 12 × 12 square Petri dishes on ½ MS +/− 50 mM NaCl. At least 100 sterilized seeds of each population were put on the media surface, stratified for 48 h at 4 °C, and then placed in a growth chamber  and under cold stress “C” (16 °C), the heat-tolerant population Herr grown at the ctrl and under heat stress “H” (28 °C), and finally the salt-tolerant population Grip grown at the ctrl and under salt stress “S”. The roots were harvested into microcentrifuge tubes containing metallic beads after 14, 10, or 8 days for plants grown at 16 °C, 22 °C, and 28 °C, respectively, and immediately frozen in liquid nitrogen.The plants were ground via RETSCH Mixer Mill MM 400 for 2 min at 30 Hertz. The RNA was extracted using the Qiagen RNeasy Plant Mini kit according to the manufacturer’s instructions and dissolved into 50 μL elution buffer. The yield of RNA was determined by taking spectrophotometric readings by a Denovix DS-11 Nanodrop spectrophotometer. RNA sequencing and the preparation of RNA libraries were performed at Genewiz in Leipzig, Germany.A. thaliana cDNA library (TAIR10_cdna_20101214_updated) in batches, one reference sequence per transcript. The mapping process was parameterized by a match score = 1, a mismatch score = 2, a linear gap cost with insertion and deletion costs = 3, length fraction = 0.95, a similarity fraction = 0.98 to maximize stringency, and a maximum number of hits per read = 10.The obtained data were treated using Qiagen CLC Genomics Workbench software . First, the sequenced reads were trimmed prior to mapping using quality-scores-based trimming , trimming of ambiguous nucleotides (maximum number of ambiguities = 2), sequencing adaptors trimming , and length trimming by which short reads less than 50 nucleotides were discarded. Then, the trimmed reads were mapped against the TPM or “Transcripts per million”, a normalization method for RNA-seq, was selected as the expression value  less than or equal to 0.05 was used to detect the significant differentially expressed genes (DEGs).The analyses of the genes differential expression (DE) were performed on the whole transcriptome using statistical differential expression tests for the set of expression tracks with the associated metadata by multifactorial statistics based on a negative binomial linear model (GLM) while assuming that the read counts follow a negative binomial distribution . The effhttps://github.com/FelixKrueger/TrimGalore) with -q 30 --length 20 options. The cleaned reads were mapped onto the TAIR10 A. thaliana reference genome Col-0 using bowtie2 (v 2.3.5.1) [p-value 0.01 –strand-filter 1 –output-vcf 1. All polymorphic sites were then identified among the populations. Finally, an SNP calling based on all accessions was performed on all polymorphic sites to differentiate null values (NA) from the reference allele. The single nucleotide polymorphisms (SNPs) identified were then located on the Col-0 genome and have been classified as (i) upstream, (ii) downstream, (iii) in the coding, or (iv) in the noncoding regions of each gene. In the frame of this study focused on CIII Prxs, only SNPs variation in those genes has been presented.Genomic DNA was extracted from a pool of individuals for each population. The rosette leaves of 3- to 4-week-old plants were harvested and frozen in collection microtubes  containing metallic beads. They were then ground for 2 min at 30 Hertz using the RETSCH Mixer Mill MM 400. The obtained powder in each well was incubated with 400 μL of a DNA extraction buffer 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, 1.25% sodium dodecyl sulfate (SDS) , 1% PVP, 1% sodium metabisulfite , 10 μg/mL RNase A , and 0.5 mg/mL proteinase K ) at 65 °C for 30 min. After cooling and centrifugation, 130 μL of 4 M potassium acetate  and 0.35 M acetic glacial acid  mixture were added and the plate was incubated at −20 °C for 30 min. The resulting supernatants were transferred into a 96-well column plate , where 600 μL of the fixation buffer  and 100% ethanol ) were added to each sample. After centrifugation, the new supernatants were transferred into a 96-well AcroPrepTM Advance filter plate  fixed above a new 96-well column plate. The plates’ block was centrifuged, and the effluents were discarded. The filters were washed twice with 650 μL of a washing buffer . Finally, DNA was eluted into an elution plate  with 50 μL of Tris-EDTA buffer added to each well. The extracted genomic DNA from 1 to 5 individuals corresponding to each population were pooled together into equimolar DNA pools that were sent for Illuminas sequencing by NovaSeq 6000 sequencer at the GeT-PlaGe GenoToul platform. The raw fastq reads were cleaned by removing the adapters and the low-quality sequences using cutadapt (v2.1)  and Trim2.3.5.1)  with -no2.3.5.1)  markdup 2O2. The total proteins from selected populations grown under contrasting conditions were extracted with 10 mM Tris Buffer , containing 20 mM EDTA , and 5% PVP . The protein concentrations were measured following Bradford’s protein quantification assay in 96-well plates . After the addition of the diluted Bradford reagent (1X) , the optical densities of the samples were recorded at 590 nm wavelength. Formerly, the spectrophotometer . To estimate the peroxidase activity, an 8:1 ratio of 200 mM phosphate buffer pH = 6 containing 0.125% guaiacol  was added to 11 mM H2O2  and incubated immediately with the protein extracts. OD470 was successively registered 1 and 2 min after the reaction started. The peroxidase-specific activity was calculated . Two-way ANOVA tests for multiple independent samples were carried out to determine the effects of the different treatments and the populations on the peroxidase activity [The peroxidase activity was assayed using guaiacol/Hactivity .A. thaliana Pyrenean populations that developed contrasted root phenotypes under thermal and saline stresses. This contrast allowed the identification of three stress-tolerant populations from the Pyrenean collection—Eaux against cold, Herr against heat, and Grip against salinity.This study benefitted from the phenotypic plasticity displayed by CIII Prxs in these stress-tolerant populations was elaborately analyzed via RNA-seq. As expected, a myriad of genes had different expressions among populations under control conditions, reflecting their genetic diversity. Additionally, the expression of these genes significantly changed because of stressful treatments. Interestingly, these changes were contrasted between the Pyrenean populations and Col-0.Afterward, the transcriptional regulation of CIII Prxs regulations. To illustrate, several CIII Prxs were exclusively upregulated in the Pyrenean populations under stressful conditions, and hence novel roles in stress tolerance were associated with these genes. Furthermore, the compilation of these comparisons upgraded the available knowledge about the functional specificity of different CIII Prxs, where particular genes were identified to be involved in response to specific stress.The treatment- and genotype-based differential expression (DE) analyses revealed additional aspects of the Indeed, the novel and specific putative roles in stress tolerance that were associated with cIII genes opened future perspectives to study tolerance mechanisms. Since such mechanisms rely on complex and interconnected regulatory pathways, further studies should integrate all biological aspects and scales from the molecule to the organism to achieve a better understanding of plant tolerance.


This study was conducted to achieve the following three aims. Firstly, to understand the solubility mechanism of granular sodium metasilicate pentahydrate (Na2SiO3.5H2O) when used in a one-part mixing method. Secondly, to investigate the properties of AAMs when a sodium metasilicate aqueous solution is used as an alkaline material and as a source of silica. Lastly, to study the retardation effect of sucrose on AAMs. This research used aluminum silicate precursors, such as low-calcium fly ash, slag, and micros silica, alkali activators, such as NaOH pellets and Na2SiO3.5H2O, and standardized sand. The alkaline activators were first dissolved in water using a water bath shaker to achieve the alkaline solution. Sucrose, which is about 2% of the weight of the solid precursors, was added to modify the reaction process between the precursors and the alkaline materials. Four types of samples were prepared: M1, M2, M3, and M4, with the fly ash, slag, and silica fume ratios of 80:20:0, 70:30:0, 75:20:5, and 100:0:0, respectively. The research conducted solubility test of the alkaline materials, flowability, 7-, 28-, 56-day compressive and flexural tests, drying shrinkage test of mortar samples, and the setting tests of pastes with and without sucrose. The results show that the dissolution time of the NaOH was much shorter, whereas Na2SiO3.5H2O needed a solvent with a temperature of around 40 °C to be fully dissolved. This problem of solubility decreases the quality of AAMs formed using the one-part mixing method. Among the mortar samples, the M4 had the highest flow rate, while M3 had the lowest flow rate. M2 had the highest compressive and flexural strength of 43.4 MPa and 6.1 MPa, respectively. The setting time test shows that sucrose retards the reaction process in AAM.This research investigates the properties of alkali-activated materials (AAMs) using sodium metasilicate, with the ratio of SiO Alkali-activated material, Sodium metasilicate, Solubility, Sucrose, Setting time test. Annual global production of ordinary Portland cement (OPC) reaches 4 billion tons . It is nmissions . The pro2 to the atmosphere [When aluminosilicate materials, such as fly ash and slag, react with an alkali source, it produces a material that has binding properties , 6. Suchmosphere , 8. Moremosphere . Besidesmosphere , 11. Furmosphere , 13, 14.2SiO3, and K2SiO3, is used as an alkali source in AAMs, as geopolymerization and activation of AAM occur in an alkaline environment. Water glass (Na2SiO3) provides such an environment [2SiO3, such as potassium silicate. However, using potassium silicate has some disadvantages, such as increased specific surface area values, a lower degree of crystallinity, and lower resistance to attack by HCL [Aqueous alkaline solution, such as NaOH, KOH, Naironment , and theironment . Moreoveironment , and sodironment . Some stk by HCL . Therefok by HCL .AAMs are made conventionally by the reaction of aluminosilicate precursors, such as fly ash and slag, and aqueous alkaline solution, such as hydroxides, silicate, and carbonate. This method for making the materials is called the two-part mixing method , 20, 21.2SiO3.5H2O), a type of sodium metasilicate, were used as alkaline activator (AA). The result showed that the strength of AAMs made using the one-part mixing method was far less than the one made by the two-part mixing method. Moreover, some other researchers have also reported that the AAMs made using the one-part mixing method had lower mechanical strength than the one made using the two-part mixing method [2SiO3.5H2O in different solvent temperatures. The result will help the further development of the method.The author of this study investigated the properties of AAM made by both one-part and two-part mixing methods and compared both results in previous unpublished research. Sodium hydroxide (NaOH) pellets and sodium metasilicate pentahydrate  of around 2, which makes the AAM viscose and causes application problems [2SiO3 is decreased when reducing the SiO2:Na2O ratio.The granular Nag method , 28, 29.g method , 31 and problems , 33, 34.problems , the visFly ash-based AAMs, especially those with a small amount of Ca+ in their composition, have a much higher setting time due to their slow reactivity. Therefore, they are cured at higher temperatures , 37. IncThe authors observed the problem of quick setting in a one-part mixed AAM mortar, which had a significant amount of slag in their composition in the previous unpublished experiment. Using sucrose delayed the solidification of the mortar, thereby making ease in the casting of the mortar. Furthermore, sucrose increased the strength of the mortar. This may be due to the modification of the reaction process caused by the addition of sucrose and better dispersion of the precursors and alkaline material throughout the material because of the improved workability. This research also studies the retardation behavior of sucrose on AAMs through setting time test since the retarding behavior of sucrose on OPC materials is reported by some researchers. Some researchers have concluded that sucrose retards the setting time and improves the microstructure of OPC concrete , 41. Bes22.12/gr, and microsilica were used as aluminum silicate precursors. Standardized sand compatible with the ISO 679:2009 was used as fine aggregate, and the sand to binder ratio was 2.97. The chemical composition of the materials is shown in 2SiO3.5H2O that consists of 30% Na2O, 29% SiO2, and 41% H2O. Sucrose, which is about 2% of the weight of the solid precursors, was added to modify the reaction process between aluminosilicate precursors and alkaline materials.Fly ash type II, ground granulated blast-furnace slag (GGBFS) with a Blaine size of 6000 cm2SiO3: NaOH) was 1.5 for all types of mortar and paste samples. The liquid to binder ratio was set to 0.33.Overall, four types of mortar samples were made in this experiment. Tables 2.22.2.1To improve the reaction process of making the AAM, fly ash, slag, and silica were premixed in a mixer called Omni mixer.An Omni mixer with a 5 L capacity and rotation speed of 2.5 Hz (2.5 spin or round per second) was used to mix the materials. The mixing duration was first set to 2 h. However, when the materials were not appropriately mixed and some white slag particles were visible, the mixing duration was increased to 3 h. After the 3-hour mixing, the materials were well mixed than before. The premixed materials were covered by plastic bags and put inside a bucket with a lid. The bucket was then covered with a lid.2.2.22SiO3.5H2O cannot easily dissolve in water at room temperature and need a solvent with a temperature of around 40 °C or above. This was found by the solubility test of the alkaline materials conducted in this study. The details and results of the experiment are explained in the result section.NaOH pellets can quickly dissolve in water at room temperature, but granular Na2SiO3.5H2O was placed inside the flasks in 3 equal groups of shaking. During the dissolution of NaOH pellets in water, the solution's temperature increased. It helped the first group of granular Na2SiO3.H2O to be well dissolved in the NaOH solution. When the temperature decreased, the granular Na2SiO3.5H2O could not be dissolved well. Therefore, in the second and third sets, the tub water temperature was increased to 40°C–45 °C, respectively.A water bath shaker was used to make an AA solution. It was equipped with a heater to control the temperature of the water inside, wires to hold the flasks, and a shaking mechanism. The making of the alkaline solution was as follows. First, the shaker's tub was half-filled with water. Then, flasks with a specified amount of distilled water were placed inside it and tightened by its wires. The temperature of the water inside the tub was set to 30 °C. The shaking rate was 120 rounds/min. A specific NaOH powder was put inside each flask in two sets, then shook for 3 min for complete dissolution. Granular Na2SiO3.5H2O dissolved entirely in NaOH solution, it was put in a larger flask and stored at room temperature. After some time, it was noticed that the solution was susceptible at normal temperature, and solution crystallization could quickly occur inside the solution. Therefore, the AA solution was kept in an incubator at 45 °C immediately after its making. After that, the crystallization did not occur, and the solution was transparent, smooth, not viscose, and looked like water.After Na2.3The experiment prepared prisms of 160 × 40 × 40 mm for both compressive and flexural strength and shrinkage tests. The materials were mixed in a paddle mixer.During the mixing procedure, as the solution's temperature decreased due to the solute crystallization, the flow and workability of the mortars decreased. Therefore, it was too difficult to cast them into molds. This mixing condition was called the ambient mixing condition.Then, it was decided to keep the mixing materials in the incubator at 45 °C for at least 24 h, and then mix the materials at a constant temperature of 24°C–26 °C. This mixing condition was named the hot mixing condition. The mixing procedure was done inside a tent at above mentioned temperature. The study observed a significant improvement in the flow and rheology of the mortar.The flowability was measured according to JIS R5201. A flow table was used to test the flowability. The mortar was put in the cone in two equal layers. Each layer was tamped 15 times by a tamped rod. Then, excess mortar material was cut off and the surface of the cone was flattened. Then, the cone was slowly lifted upward. The plate was then repeatedly risen and dropped 15 times. Next, the mortar was spread. The maximum direction and the opposed direction, which was perpendicular to the maximum direction were measured. This procedure was done twice and the average value in millimeter (mm) was taken as the flow value of the mortar. After that, the mixtures were poured inside prisms and vibrated for 2 min. To avoid crystallization of the solutes inside the mix and facilitate the reaction process between aluminum silicate precursors and alkaline solution, the prisms were put at 45 °C and 94% relative humidity inside an incubator for two days. After that, they were cured in the control room at 18°C–20 °C and 30%–50% relative humidity.In this research, the solubility test of the solid alkaline materials, flow test, 7-, 28-, 56-day compressive and flexural strength test, and drying shrinkage of mortar samples were studied. Moreover, to investigate the retarding effect of sucrose on AAM, the mixed proportion of the raw precursors of M2, which has the highest amount of slag, and M4 were chosen to make pastes for studying the initial and final setting time. The pastes were studied in the presence and absence of 2% sucrose.33.1The reaction between aluminosilicate precursors and alkaline materials is vital for producing geopolymers and AAMs. Therefore, in a one-part mixing method, solid alkaline materials, such as NaOH and sodium metasilicate need to be well dissolved to produce the desired AAM.2SiO3.5H2O, and water was put inside a flask. The Na2SiO3.5H2O: NaOH ratio was set to 1.5. A water bath shaker was used to mix the materials. The shaking rate was set to 120 rounds/min.A solubility test of the materials was conducted to better understand the alkaline materials’ solubility mechanism in the one-part mixing method. One batch mixing amount of NaOH pellets, granular Na2SiO3.5H2O were put inside the flask and shook for 5 min. The duration of shaking was chosen according to the estimated mixing timing of making mortar or concrete in a mixer. After 5 min of mixing, the temperature of the solution increased to about 34 °C. As the dissolution of NaOH in water is an exothermic reaction, it rose the temperature of the solution to about 8 degrees Celsius. Most particles could not be dissolved in water, and those particles were the sodium metasilicate since NaOH quickly dissolved in water. For confirmation, a similar experiment was performed, in which it took less than 5 min for the NaOH to be fully dissolved in water.The temperature of the water before mixing with the alkaline materials was 26 °C. NaOH pellets and granular NaTherefore, the solution was mixed for two more minutes, but there was still no improvement observed in the dissolution rate, and the temperature of the solution decreased to 31 °C. Therefore, to increase the temperature of the solution, the temperature of the shaker's tub water was set to 35 °C and then to 40 °C, and each was mixed for 5 min. Thus, the temperature of the solution increased to 32 °C and 36.5 °C, respectively, and a significant improvement in the dissolution of the sodium metasilicate was observed. However, still some particles remained undissolved. When the temperature of the solution increased to 41 °C, total dissolution of the particles occurred.2SiO3.5H2O by a solvent with a lower temperature is difficult, and it could not be fully dissolved when adopting the one-part or the drying mixing method to make geopolymers and AAMs. For making 32% sodium metasilicate solution, a temperature of around 40 °C is required for the solvent to dissolve the solid material thoroughly. Concerning increment of some degrees of temperature by NaOH, a solvent with a temperature slightly lower than 40 °C can also be enough for the solubility of the sodium metasilicate. The temperature of the aluminum silicate precursors and aggregates may also influence the solubility of the materials. Therefore, the hot mixing condition will have a significant effect on the one-part mixing method. Moreover, besides maintaining the proposed temperature, increasing the mixing duration and reducing the Na2SiO3.5H2O: NaOH ratio may also play a significant part in the dissolution of the material in the one-part mixing method.This study shows that the dissolution of the granular Na3.2The flowability test was conducted to observe the influence of the aluminosilicate precursors and the change in the mixing conditions on the flow of the samples. Moreover, the flow and rheology of the alkaline solution formed by the sodium metasilicate are discussed in this section.2SiO3.5H2O was smooth and fluent and there was no sign of viscosity. But as mentioned earlier, the alkali solution was very sensitive to the temperature variation as the crystallization of the AA solution occurred at a temperature lower than 30 °C. Therefore, the workability and flowability of the mortars were decreased at the aforementioned temperature. The same phenomena of crystallization of the water glass or sodium silicate can also happen while adopting the one-part mixing method using granular Na2SiO3.5H2O. The crystallization in the solution may reduce the reaction process between the alkali and the aluminosilicate precursors. Hence, the properties of the AAMs formed can be reduced.The alkaline solution formed by NaOH pellets and granular NaIn this study, it was decided to change the temperature of the materials and the mixing condition. All materials were kept at 45 °C for 24 h, and mixing was done at a controlled temperature of 24°C–26 °C. When the mixing condition was changed to the hot mixing condition, significant improvement in the workability of fresh mortar was observed and crystallization did not occur.Before applying the hot mixing condition, the M1 sample was mixed in an ambient mixing condition, and the flow of the sample was so low that it was not easily put inside the molds. The flow value of the M1 was 102.5 mm. There was about a 15% increment in the flow value of the mixture when the hot mixing condition was applied. A higher flow value was achieved due to a reduction in the mix's cohesiveness at a higher temperature.Among the samples, the M4 sample had the highest flowability, and its flow value could not be measured due to its overflow from the flow table. It was due to the low reactivity of low-calcium fly ash or fly ash type II  which caPrevious studies , 34, 43 3.3Compressive and flexural strength are an important characterization of AAM. As the sodium silicate improves the reaction process between the precursors and alkali , so the Slag had a significant influence on both the compressive and flexural strength of the samples due to the presence of a high amount of Ca + that increases the reactivity of the material . The M2 The M4 sample, made of 100% fly ash, had the lowest compressive strength of 17.8, 20.6, and 21.2 MPa at the ages of 7, 28, and 56 days, respectively. The lowest compressive strength for all ages was due to the extremely low reaction between the aluminosilicate precursor and alkaline solution.Following the compressive strength result, the M2 sample had the highest 7-, 28-, and 56-day flexural strength, which is approximately 6 MPa for all ages, followed by the M1 and M3 samples, which both had the flexural strength of approximately 5 MPa for all ages. The M4 sample had a flexural strength of 3.7, 4.2, and 4.7 MPa at the ages of 7, 28, and 56 days, respectively.All samples were cured at 45 °C and 94% relative humidity inside an incubator for two days. Afterward, they were cured at 20 °C and 30%–50% relative humidity. Therefore, they gain most of the strength within a short time. The buildup in strength was due to the increase in the reaction or hydration rate due to heat and steam inside an incubator , 45, 46.Compressive strength of more than 40 MPa and flexural strength of 6 MPa were achieved using 32% concentrated sodium metasilicate solution, which is much lower than the concentration of sodium silicate solution used in making traditional geopolymer and AAMs , 48, 49.3.4Drying shrinkage of the samples was studied according to . The tesThe M1, M2, and M3 samples had higher amounts of shrinkage compared to M4 due to the presence of the slag and silica fume in their composition, which caused the quick reaction and early loss of water in the mixture. The shrinkage of the samples was increasing until 50 days. After that, it was decreased and then flattened. In contrast, the shrinkage of the 100% fly ash sample was nearly 0 microstrain at the age of around 90 days. This is attributed to the slow reactivity of the precursor . The dryThe shrinkage of all samples was below 0.07%, but it was within the range of allowable shrinkage defined by the ACI 209R, which is 0.05%–0.078% . This ma3.5The setting time test was conducted to understand the retarding influence of sucrose on AAMs. The initial and final setting times of P2 and P4 were studied according to JIS R 5201. The mixed proportion of aluminosilicate precursors of M2 and M4 was used to make pastes, which are named P2 and P4, respectively. The liquid to the binder ratio was kept to 0.19 and 0.17 for the P2 and P4 pastes, respectively. Figures The P4 pastes, P4S0% and P4S2%, followed the same trend: sucrose increased the initial setting time of P4 from 310 to 515 min and the final setting time from 433 min to 990 min.The initial and final setting time test results of the pastes revealed that the retarding behavior of sucrose can also be observed on AAMs. Many researchers have confirmed its retarding effect on OPC materials in the past , 41. The41)2SiO3.5H2O increases with an increase in temperature. Total dissolution of the material for making 32% sodium metasilicate solution occurs at a temperature of around 40 °C. If the aforementioned type and concentration of water glass or sodium silicate are changed, then a different temperature of a solvent will be required to totally dissolve the material. Therefore, it is essential to check the solubility of sodium silicate before using it in a one-part mixing method.The dissolution of granular Na2)2 and concentration of sodium metasilicate solution. However, the alkaline solution is more sensitive to the lower temperature. Crystallization of the solution can easily happen at a temperature below 30 °C. The same phenomena of crystallization of water glass can also occur while adopting a dry mixing method or one-part mixing method. This crystallization can reduce the reaction process between alkali and aluminum silicate precursors that result in reduction in the quality of the alkali-activated product. To avoid crystallization and to keep the solution smooth and fluent, storing it at a higher temperature is recommended.The alkaline solution formed by the sodium metasilicate is more fluent and smoother. This is due to the lower amount of SiO3)A compressive strength of around 40 MPa can be achieved by combined fly ash and slag-based AAM activated by a low concentrated sodium metasilicate and NaOH solution.4)2:Na2O = 2) sodium silicate solution.Using sodium metasilicate solution in the two-part mixing method can solve the viscosity problem created by a higher modulus (SiO5)Sucrose retards the reaction process between aluminosilicate precursors and AA solutions in AAM, hence, the setting time is delayed.One-part mixing method was studied by many researchers to make ease in the production of AAMs and geopolymers and to generalize the materials. None of the researchers checked the solubility of the alkaline materials used in the aforementioned mixing method. Therefore, this research studied the dissolution mechanism and solubility of the alkaline materials used in a one-part or dry mixing method. Moreover, the aqueous solution from sodium metasilicate and NaOH was made to overcome workability and rheological problems. Furthermore, the retardation behavior of sucrose on AAM was studied by setting time test. This study concludes as follow:Mohammad Idris Rasuli: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Yuyun Tajunnisa: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data.Akifumi Yamamura: Performed the experiments.Mitsuhiro Shigeishi: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data.10.13039/501100012009Hitachi Global Foundation Ref. RS-15, E-1 March 8, 2019, 10.13039/501100004091Kumamoto University, and 10.13039/501100013355Institut Teknologi Sepuluh Nopember.This work was supported by the Data related to our research is not publicly available at this time, but will be deposited in a publicly accessible repository after the dissertation, including the contents of this thesis, is reviewed.The authors declare no conflict of interest.No additional information is available for this paper.


The human body requires energy to function. Adenosine triphosphate (ATP) is the cellular currency for energy-requiring processes including mechanical work . ATP used by the cells is ultimately derived from the catabolism of energy substrate molecules—carbohydrates, fat, and protein. In prolonged moderate to high-intensity exercise, there is a delicate interplay between carbohydrate and fat metabolism, and this bioenergetic process is tightly regulated by numerous physiological, nutritional, and environmental factors such as exercise intensity and duration, body mass and feeding state. Carbohydrate metabolism is of critical importance during prolonged endurance-type exercise, reflecting the physiological need to regulate glucose homeostasis, assuring optimal glycogen storage, proper muscle fuelling, and delaying the onset of fatigue. Fat metabolism represents a sustainable source of energy to meet energy demands and preserve the ‘limited’ carbohydrate stores. Coordinated neural, hormonal and circulatory events occur during prolonged endurance-type exercise, facilitating the delivery of fatty acids from adipose tissue to the working muscle for oxidation. However, with increasing exercise intensity, fat oxidation declines and is unable to supply ATP at the rate of the exercise demand. Protein is considered a subsidiary source of energy supporting carbohydrates and fat metabolism, contributing to approximately 10% of total ATP turnover during prolonged endurance-type exercise. In this review we present an overview of substrate metabolism during prolonged endurance-type exercise and the regulatory mechanisms involved in ATP turnover to meet the energetic demands of exercise. Humans are capable of performing extraordinary feats; with both natural endowment and sport-specific training, athletes can perform various activities with astonishing power, speed, skill and tolerance . Even foResearch into exercise metabolism and the substrates necessary to support different activities has been extensive and spans just over a century ,23,24,25In exercise physiology, exercise can be broadly characterized as either low load, high repetition (endurance activities) or high load, low repetition (weight or resistance training) ,41, and −1 [−1 [2max lasting >45 min, while fat stores are abundant in the body  and may theoretically support many hours of exercise at the same intensity [Endogenous carbohydrates are mostly stored as glycogen in the skeletal muscle and liver ,48. Skel−1 . Manipul−1 [−1 . Glycogentensity ,44,53.2+) release and an adrenaline-mediated increase in cyclic AMP (cAMP), thereby activating phosphorylase kinase (PK) and the resultant activation of glycogen phosphorylase [Muscle glycogen degradation depends on the activation of glycogen phosphorylase and debranching enzymes, which ultimately split glucose residues from the glycogen chain . Activathorylase ,57.Muscular contraction also activates AMP-activated protein kinase (AMPK) by an increase in the cellular AMP/ATP ratio when there is a drain on ATP . AMPK is2+ signal into a cellular process [2+ concentration in the cells, via activation of phosphorylase kinase, triggers glycogen degradation [Calmodulin, the δ-subunit of phosphorylase kinase, associates with the protein troponin that stimulates skeletal muscle contraction . It is a process . An incrradation ,66.An acute exercise bout’s intensity and duration predominantly influences muscle glycogen degradation rates. As a consequence, muscle glycogen degradation exponentially increases with exercise intensity and, over time, progressively declines due to the finite nature of this carbohydrate store within skeletal muscles . AnotherThe mode of exercise and whether or not carbohydrates were ingested during exercise seem to be interrelated determinants of equal importance on utilising muscle glycogen during physical work . For exaSex differences may play a role in the discrepancies in the utilisation of endogenous carbohydrate stores ,83. It h2max and estimated physical activity level can only account for 34% in the variability in peak fat oxidation and thus the inter-individual variation in fat oxidation remains largely unexplained [Training status affects the selection of substrate use during exercise as a result of an enhancement in lipid oxidation and a concurrent improved insulin-stimulated glucose uptake capability when compared with untrained individuals ,89,90. Explained . Howeverxplained . Other fxplained ,95,96,972max) exercise intensities, whereby splanchnic glucose production was sufficient to deplete 75% of the liver glycogen stores [Although muscle glycogen plays a central role in energy metabolism during moderate to high intensity exercise, the importance of other extra-muscular carbohydrate sources  is profound when performing prolonged exercise . The remn stores . These tn stores . The livn stores , which in stores  and physn stores . The impn stores  and a sun stores .In healthy individuals, the liver ensures the maintenance of glycaemia within a tight range under widely divergent physiological conditions. This is achieved through the dynamic equilibrium of hepatic glucose production mechanisms (namely glycogenolysis and gluconeogenesis) by which the relative contribution is determined by the intensity and duration of exercise, in addition to the absorptive state of subjects ,104. UndSimilarly, during exercise, the contribution of both glycogenolysis and gluconeogenesis cannot be overstated in the absence of nutrient (carbohydrate) ingestion. In fact, an appreciable amount (15–30%) of the energy required for moderate exercise is obtained from blood glucose , while s2max, [Glucose output is almost entirely derived from liver glycogenolysis at the initial stages of prolonged moderate to high-intensity exercise secondary to glucagon and noradrenaline-mediated activation of hepatic glycogen phosphorylase ,102. Ind2max, . The latGlucoregulation is accomplished by a combination of regulatory controls, namely through feedback  and feed2max) endurance exercise [2max, while 10% of lactate was indirectly oxidised via gluconeogenesis [Lactate is a crucial intermediate in energy metabolism and it is known to be produced in many tissues including the skeletal muscles and liver . Lactateexercise . Most laexercise ,122,123.exercise ,124. Thiexercise ,126. Recogenesis . The metogenesis ,120. Durogenesis ,128. In ogenesis . An accuogenesis .Fat oxidation is the second dominant substrate for endurance exercise ,130. AltThe use of fat as an energy substrate involves hydrolysis of triacylglycerols (TG) to FA and glycerol , and the delivery of the released FA for oxidation in skeletal muscle mitochondria. TG can be derived from three sources: adipose tissue TG, intra-myocellular TG (IMTG) and circulating plasma TG . During 2max) the contribution of fat oxidation is blunted by ≈34% compared with fat utilisation rates during lower intensities (≈55% VO2max) [2max) becomes down-regulated compared with moderate intensities (≈65% VO2max) [TG may practically serve as an unlimited energy source to support the energetic requirements during endurance exercise, given their quantitative superiority to be stored over carbohydrates, and their higher energy density than carbohydrates . On the  VO2max) . In a si VO2max) . The sup VO2max) .A number of factors have been suggested for the down-regulation of fat oxidation at higher exercise intensities. Failure in adipose tissue to supply the exercising muscle with sufficient FA may be related to an inhibition of fat mobilisation or an inadequate perfusion of the adipose tissue . SecretiWhen focussing on the limitations of FA oxidation from the transition from moderate to high-intensity exercise, the failure of the muscle to oxidise FA in the mitochondria may also be a candidate. The oxidation of long chain FA  have to be converted to their acylcarnitine form to enter the mitochondria for β-oxidation by a reaction catalysed by carnitine palmitoyltransferase 1 (CPT-1) . Indeed,2max [Malonyl-CoA is an intermediate in the de novo synthesis of FA and is an allosteric inhibitor of CPT-1, thereby inhibiting fat oxidation . It is f2max , with gr2max , althoug2max . At high2max ,142.Protein metabolism serves as an auxiliary fuel source during prolonged endurance exercise by contributing to ≤5% of ATP provision . Althoug2max) leucine oxidation and BCOAD activation were lower in both males and females [2max [During exercise, the branched-chain amino acids (BCAA) are preferentially oxidised to other forms of amino acids through a transamination process to become transferred into keto-acids by the rate-limiting enzyme branched-chain oxo-acid dehydrogenase (BCOAD) ,172. The females . However females ,171. Thees [2max  and thates [2max .The role of protein metabolism during exercise has also been linked to the provision of precursors for tricarboxylic acid (TCA) reaction; a major common pathway for the oxidation of carbohydrates, fats and amino acids ,181. TheAlanine is a dispensable amino acid that can be synthesized endogenously by the liver, acting as an subsidiary source of energy during extreme circumstances such as starvation and prolonged endurance exercise . This prGlycerol is a hydrophilic carbon skeleton of sugar alcohol that can be easily transported as a free molecule in the bloodstream in response to triglyceride hydrolysis and FA mobilization. Lipolytic activity within adipose tissue and intramuscular fat is highly active during moderate-intensity endurance exercise by the action of catecholamines, leading to elevated circulating glycerol levels ,188. GlyKetone bodies are lipid-derived low molecular weight hydrophilic molecules, and are normally found in circulation in relatively small quantities ranging between 0.1 to 0.5 mmol/L . They arAfter over a century of research, the regulation of metabolism in endurance-type activities remains an area of interest for exercise physiologists, biologists and nutritionists ,198,199.Endurance exercise improves overall health and enhances sport performance through a shared mechanism of increased metabolic activity in response to a high energetic and/or oxygen demand of the contracting skeletal muscles; as a result, different bodily systems respond in an integrative fashion to meet this homeostatic challenge. If said exercise stimulus is repeated over time, chronic structural and functional changes occur ,200. Ind