extfieldsofstudy: Medicine Biology

text:
Methyljasmonate and salicylic acid contribute to the control of Tilletia controversa Kühn, causal agent of wheat dwarf bunt

Tilletia controversa Kühn (TCK) is the causal agent of dwarf bunt of wheat, a destructive disease in wheat-growing regions of the world. The role of Meja, SA and Meja + SA were characterized for their control of TCK into roots, coleoptiles and anthers. The response of the defence genes PR-10a, Catalase, COI1-1, COII-2 and HRin1 was upregulated by Meja, SA and Meja + SA treatments, but Meja induced high level of expression compared to SA and Meja + SA at 1, 2, and 3 weeks in roots and coleoptiles, respectively. The severity of TCK effects in roots was greater at 1 week, but it decreased at 2 weeks in all treatments. We also investigated TCK hyphae proliferation into coleoptiles at 3 weeks and into anthers to determine whether hyphae move from the roots to the upper parts of the plants. The results showed that no hyphae were present in the coleoptiles and anthers of Meja-, SA- and Meja + SA-treated plants, while the hyphae were located on epidermal and sub-epidermal cells of anthers. In addition, the severity of hyphae increased with the passage of time as anthers matured. Bunted seeds were observed in the non-treated inoculated plants, while no disease symptoms were observed in the resistance of inducer treatments and control plants. Plant height was reduced after TCK infection compared to that of the treated inoculated and non-inoculated treatments. Together, these results suggested that Meja and SA display a distinct role in activation of defence genes in the roots and coleoptiles and that they eliminate the fungal pathogen movement to upper parts of the plants with the passage of time as the anthers mature.

Results
PR-10a, catalase and CoI1-1 transcriptional levels in roots after treatment of wheat plants with resistance inducers. The transcriptional level of PR-10a was analysed by qPCR in the roots of a highly susceptible cultivar (Dongxuan 3) after treatment with inducers of resistance (SA, Meja and Meja + SA). For PR-10a, Meja induced the maximum level of expression at 1 week with a 12.26-fold increase in treated non-inoculated (inducer) plants compared to reference. In treated inoculated (inducer + TCK) plants, an 8.98fold increase was recorded after Meja treatment, but in the non-treated inoculated (TCK) group, an increase of only 1.39-fold was recorded after treatment with Meja. SA treatment downregulated the expression of PR-10a by 0.18-fold (inducer + TCK) and 0.45-fold (inducer) at 1 week compared to the reference (Fig. 1a). A similar response was recorded after 2 and 3 weeks for Meja and SA (Fig. 1b,c). With regard to Meja + SA, the maximum transcription level of PR-10a was noted at 3 weeks, reaching a 4.29-fold increase compared to the reference in inducer (Fig. 1c), and it reached a 3.84-fold increase compared to the reference at 1 week (Fig. 1a). The above results indicated that the expression of PR-10a was increased at 1 week in the Meja treatment compared to the expression level at 2 and 3 weeks, which may be due to the prevalence of TCK in the roots during the first week compared to the other weeks. The inducers of resistance may have degraded the cell wall of TCK during weeks 2 and 3, or they may have activated the host resistance genes or pathogen avirulence genes, encoding specific elicitor molecules.
The response of catalase to the exogenous application of hormones was comparatively higher compared to the reference. As shown in Fig. 1, catalase expression slowly increased over time. The expression level of catalase increased to 50.95-fold (inducer) after 3 weeks of SA treatment (Fig. 1f), and significant expression was recorded after 1 week and 2 weeks in the inducer and inducer + TCK plants, respectively, for SA (Fig. 1d,e). Similarly, catalase responded in a similar way to the exogenous application of Meja at 3 weeks. The highest expression in the Meja treatment occurred at 3 weeks with an increase of 26.9-fold for inducer plants, which was higher compared weeks 1 and 2 (Fig. 1f). In the Meja + SA treatment, upregulated expression of catalase was recorded in weeks 2 and 3 (Fig. 1e,f), but the catalase expression was reduced in the inducer + TCK and inducer treatments compared to the reference at 1 week (Fig. 1d).
COI1-1 responded similarly as it was downregulated by both Meja and SA after 1 week in all treatments, but a significant upregulation in COI1-1 was noted in the TCK treatment compared to the reference at week 2. The expression of COI1-1 was significantly upregulated after Meja + SA treatment at week 1 in the inducer treatment compared to the reference (Fig. 1g). With regard to Meja, COI1-1 expression was increased by 4.89-fold and 2.59-fold in the inducer + TCK and inducer treatments, respectively, compared to the reference (Fig. 1i). In the SA treatment, COI1-1 expression was increased by 2.03-fold and 1.77-fold in the inducer + TCK and inducer, respectively, compared to the reference. After the Meja + SA treatment, COI1-1 expression was increased by COI1-2 and HRin1 levels in roots and PR-10a level in coleoptiles after treatment of wheat plants with resistance inducers. COI1-2 levels were similarly upregulated by Meja, SA and Meja + SA after treatment for 1, 2 and 3 weeks in the inducer + TCK, inducer and TCK groups compared to the reference group ( Fig. 2a-c). In the 1 week Meja treatment, the transcriptional levels of COI1-2 were 7.9-fold and 5.81fold higher in the inducer + TCK and inducer plants, respectively, compared to reference. After 1 week, the COI1-2 levels were increased by 5.82-fold and 6.10-fold in the SA and Meja + SA treatments, respectively, in the inducer plants compared to reference (Fig. 2a). After 2 weeks of treatment, the highest transcriptional level of COI1-2 was in the Meja + SA treatment with compared increases of 1.24-fold, 9.8-fold and 10.79-fold in the inducer + TCK, inducer and TCK groups, respectively, compared to reference (Fig. 2b). However, the highest expression level of COI1-2 occurred after 3 weeks of treatment compared to treatments for 1 and 2 weeks. In the inducer + TCK and inducer groups after 3 weeks of Meja treatment, the COI1-2 levels increased by 14.65-fold and 7.74-fold, respectively, compared to the reference group (Fig. 2c). There was no statistically significant difference in COI1-2 expression in the inducer + TCK and TCK groups after SA application compared to the reference, but the COI1-2 expression level was 3.99-fold and 3.73-fold higher in the inducer groups after treatments with SA and Meja + SA, respectively, compared to the reference (Fig. 2c). The HRin1 expression levels after treatment for 1, 2 and 3 weeks in the inducer + TCK, inducer, TCK and reference groups were also analysed ( Fig. 2d-f). After treatment with Meja for 1 week, the HRin1 expression levels in the inducer + TCK, inducer and TCK groups were increased by 0.72-fold, 4.27-fold and 2.92-fold, respectively, compared to the reference. There was no statistically significant difference between the Meja + TCK group and the reference group. The HRin1 expression level after SA treatment decreased in the inducer + TCK and inducer groups compared to the reference. For the Meja + SA treatment, HRin1 expression levels were upregulated in the inducer treatment and TCK treatment (Fig. 2d). After SA treatment for 2 weeks, the expression of HRin1 increased by 17-fold in the inducer compared to the reference. Similarly, HRin1 expression was increased by 4.11-fold in the inducer treatment after Meja + SA application compared to the reference (Fig. 2e). After Meja treatment for 3 weeks, HRin1 expression was increased by 8.23-fold and 3.9-fold in the inducer + TCK and inducer groups, respectively, compared to the reference. After SA application for 3 weeks, HRin1 expression increased by 2.92-fold and 3.12-fold in the inducer + TCK and inducer treatments, respectively, compared to www.nature.com/scientificreports/ the reference. Moreover, after Meja + SA application for 3 weeks, HRin1 expression was increased by 3.39-fold and 4.96-fold in the inducer + TCK and inducer treatments, respectively, compared to the reference (Fig. 2f).
The PR-10a expression in coleoptiles after treatment with Meja and Meja + SA was generally upregulated in the inducer + TCK and inducer groups compared to the reference. After SA application, the expression of PR-10a was downregulated in the inducer + TCK group compared to the reference, and no significant difference was found in the TCK treatment compared to the reference (Fig. 2g). While Meja induced the highest PR-10a expression at 2 weeks, reaching increases of 5.46-fold and 3.78-fold in the inducer + TCK and inducer treatments, respectively, compared to the reference, the PR-10a expression in the TCK group was decreased by 0.71-fold compared to the reference. Moreover, a low level of PR-10a expression was detected after SA and Meja + SA treatments at 2 weeks (Fig. 2h). The PR-10a expression was high after treatment with Meja for 3 weeks in the inducer + TCK treated plants compared to the reference. There was no significant difference in the inducer and TCK groups compared to the reference group after treatment with Meja and Meja + SA. Additionally, no significant change in expression was observed in all groups after treatment for 3 weeks with SA and TCK compared to the control (Fig. 2i). COI1-1, COI1-2 and HRin1 transcriptional levels in coleoptiles after treatment of wheat plants with resistance inducers. COI1-1 expression showed a clear response to Meja in the inducer + TCK and inducer groups after 1 week with increases of 4.64-fold and 1.84-fold, respectively, compared to the reference. Compared to the reference, COI1-1 expression was downregulated after SA application for 1 week, but there was no significant change in COI1-1 expression after Meja + SA treatment for 1 week (Fig. 3a). Compared to the reference, Meja upregulated COI1-1 expression in the inducer + TCK and inducer groups by 11.39-fold and 5.24-fold, respectively, at 2 weeks (Fig. 3b). After 3 weeks of treatment with Meja and Meja + SA, COI1-1 expression was increased by 2.62-fold and 3.82-fold in the inducer + TCK treatments, respectively, compared to the reference, and there was no significant difference in SA application in the inducer + TCK and inducer treatments compared to the reference group (Fig. 3c).
COI1-2 expression in the coleoptile was upregulated by Meja and Meja + SA after treatment for 1 week in the inducer + TCK and inducer treatments compared to the reference (Fig. 3d). COI1-2 expression was increased by 3.25-fold after Meja treatment for 1 week in the in the inducer + TCK group compared to the reference. Similarly, COI1-2 expression was increased by 6.77-fold and 3.74-fold after treatment with Meja + SA for 1 week in the inducer + TCK and inducer groups, respectively, compared to the reference. With regard to SA application, there was no significant difference in COI1-2 expression when comparing the inducer group to the reference group. However, downregulated COI1-2 expression was observed in the inducer + TCK group compared to the reference www.nature.com/scientificreports/ ( Fig. 3d). A low level of COI1-2 expression was noted for Meja and SA after 2 weeks in the inducer + TCK and inducer groups compared to the reference, and the expression level of COI1-2 was downregulated in the TCK group compared to the reference after treatment with Meja and SA. No statistically significant difference was present after treatment with Meja + SA for 2 weeks in all groups (Fig. 3e). However, downregulation of COI1-2 was observed after SA application for 3 weeks compared to the reference, and no statistically significant change in COI1-2 expression was observed after Meja treatment. However, COI1-2 expression reached a peak after treatment with Meja + SA for 3 weeks with increases of 6.42-fold and 2.87-fold in the inducer + TCK and inducer plants, respectively, compared to the reference (Fig. 3f). HRin1 expression in the coleoptile was slightly upregulated by Meja treatment and significantly upregulated by the Meja + SA treatment but downregulated by SA treatment in the inducer + TCK and inducer groups. HRin1 expression in the inducer + TCK and inducer groups was increased by 2.23-fold and 2.51-fold, respectively after treatment with Meja compared to the reference. Treatment with Meja + SA in the inducer + TCK and inducer groups increased HRin1 expression by 10.86-fold and 3.55-fold, respectively, compared to reference (Fig. 3g). After treatment for 2 weeks, maximum relative expression of HRin1 was noted after treatment with SA compared to treatment with Meja and Meja + SA. SA treatment increased HRin1 expression by 7.91-fold and 9.62-fold in the inducer + TCK and inducer groups, respectively, compared to reference. Compared to reference, Meja treatment increased HRin1 expression by 6.18-fold in the inducer + TCK group, but treatment with Meja + SA increased HRin1 expression by only 2.05-fold (Fig. 3h). After 3 weeks, no statistically significant change in HRin1 expression was present after treatment with Meja treatment, but HRin1 expression was downregulated by SA treatment in all groups. However, HRin1 expression was increased by 4.17-fold after treatment with Meja + SA in the inducer + TCK group compared to the reference (Fig. 3i).
The above relative expression results showed that the inducers of resistance upregulated the expression levels of defence genes to protect against TCK after 1, 2 and 3 weeks. However, the expression changes were not consistent for specific periods of time. For example, Meja showed the best results at 1 week for one gene but not for all genes compared to SA and Meja + SA. Comparatively, inducers of resistance increased the expression level of defence genes in highly susceptible cultivars, suggesting that TCK, a bunt pathogen, can be controlled by the application of different inducers of resistance. More importantly, the overall expression levels in coleoptiles were low compared to roots, indicating that inducers of resistance degrade the fungal hyphae into roots and hyphae, thus preventing movement into the upper parts of plants. www.nature.com/scientificreports/ Hyphae development in wheat roots and coleoptiles in inoculated, non-inoculated and treated inoculated plants. To track the hyphae in roots, roots treated with different inducers of resistance were analysed by confocal microscopy. In general, we observed a gradual decrease of fungal colonization and propagation with time intervals (Fig. 4a-c). At germination, hyphae started from small tips, and they formed a hyphal network on and inside the roots. Hyphae moved into sub-epidermal cells through intercellular spaces where they branched and continued to grow. Sub-epidermal hyphae penetrated spaces between primary cell walls and plasma membranes of cortical cells. Hyphae penetrated epidermal cells and reached root cortical cells in the treated inoculated (TCK + inducers) and inoculated control (only TCK) plants, but there was no prevalence of hyphae into the non-inoculated plants. However, cortical cells were attacked more harshly in the inoculated control plants (Fig. 4a). These results showed that Meja, SA and their combination (Meja + SA) played pivotal roles in the control of T. controversa hyphae movement into the roots 1 week post-inoculation of the inducers of disease resistance. At 2 weeks, similar results were observed in all treated, inoculated control plants, but the hyphae were weaker than they were at 1 week (Fig. 4b).
To confirm these observations, infected coleoptiles were stained with WGA-AF 488 at week 3 to diagnose the presence or movement of hyphae from roots to the upper parts of plants. The confocal results showed that there was no prevalence of hyphae movement into coleoptiles in the treated plants. Moreover, some broken hyphae were observed in coleoptiles after SA treatment, but fungal hyphae were present in the inoculated control (Fig. 4c). Therefore, we tracked T. controversa into different subcells of the anthers using confocal microscopy in inoculated control and treated inoculated plants.

Hyphae proliferation into epidermal and sub-epidermal cells of anthers in non-treated inoculated plants.
To investigate the effect of T. controversa on anther cells, anthers were stained with WGA-AF 488 and PI to determine the establishment and proliferation of fungal hyphae in anther cells. Hyphae were observed on epidermal (EPI) and sub-epidermal cells, including the endothecium (EN), middle layer (ML) and pollen mother cells (PMCs) (Fig. 5a-l). Additionally, we investigated hyphal severity on EPI cells at different time intervals in non-treated inoculated plants. The results showed that at 6-and 7-days post inoculation (dpi), hyphae were observed on anther EPI cells (Fig. S1a-f and Table 1). The intensity of the hyphae increased with time, and the maximum fungal intensity on EPI cells was recorded at 8, 9 and 10 dpi (Fig. S1g-o and Table 1).

Inducers of resistance eliminate T. controversa from anther cells.
To track the hyphae of T. controversa in treated inoculated and inoculated control plants, fungal penetration into the EPI, EN and PMCs (anther cells) was analysed by confocal laser scanning microscopy (Table 1). No hyphae was observed in these three cell types (Fig. 6a-i), but highly infestation of hyphae was observed in the inoculated control anthers (Fig. 6j-l). These results indicated that there was no T. controversa hyphae penetration into the EPI, EN and PMCs of anthers after treatment with Meja ( Fig. 6a-c). Similarly, in SA and Meja + Sa treatments, no T. controversa hyphae penetration into the EPI, EN, and PMCs was observed (Fig. 6d-i). However, in inoculated control anthers, hyphae encased the EPI, EN and PMCs, resulting in cell shrinkage and rupture (Fig. 6j-l). Additionally, the physical and morphological structures of the EPI, EN and PMCs after treatment with Meja, SA and Meja + Sa indicated healthy cells compared to inoculated control anthers. These findings revealed that inducers of resistance played pivotal roles in inhibiting the penetration of T. controversa into epidermal and sub-epidermal cells of the highly susceptible cultivar of wheat.

Inducers of resistance enhance wheat resistance to T. controversa.
To further investigate the diseased symptoms in treated inoculated, non-treated inoculated and control plants, we observed three seeds from spikes of each treatment. The results TCK hyphae were not present in the seeds from the treated inoculated and control plants ( Fig. S2a-d), while clear and obvious bunted seeds were found in the non-treated inoculated plants (Fig. S2e). These data suggested that inducers of resistance contribute to control the dwarf bunt disease of wheat. Furthermore, we compared the treated inoculated, non-treated inoculated and control seeds collected from the spike of every treatment to investigate the symptoms in more detail. Normal seeds were observed in treated inoculated and control plants (Fig. S3a,c,e,g), while bunted seeds with clear symptoms were observed in non-treated inoculated plants (Fig. S3b,d,f,h). Additionally, we cut the seed longitudinally to compared the black powder inside the seeds in the non-treated inoculated plants to the treated inoculated and control plants. There were millions of teliospores in the seeds collected from non-treated inoculated spikes (Fig. S4b,d,f,h), while a white-like powder was observed in the treated inoculated and control seeds (Fig. S4a,c,e,g). Plant height is parameter affected by TCK, and stunted growth was observed in the infected fields, indicating that TCK-infected plants showed stunted growth compared to treated inoculated and control treatments (Fig. 7).

Discussion
Wheat is one of major staple food crops worldwide. However, yield losses of wheat due to T. controversa under favourable conditions can reach up to 70-80% in some wheat growing areas. Controlling dwarf bunt disease of wheat by resistant cultivars is a laborious and time-consuming process. Here, we reported the role of inducers of resistance to control dwarf bunt in the roots and coleoptiles, and we analysed the infection mechanism of TCK in anther epidermis and sub-epidermal cells. Assays using confocal microscopy, qRT-PCR and physiological traits showed that inducers of resistance (i.e., Meja, SA and combination of Meja + SA) enhanced resistance to dwarf bunt disease in a highly susceptible wheat cultivar. Overall, the data demonstrated that inducers of resistance are important mediators that increase the defence mechanism of wheat against T. controversa. New environmentally friendly molecules to protect against wheat biotic stress should be investigated to broaden insights on abiotic and biotic stress signalling pathways in different plant species. In this experiment, exogenous application of Meja, SA and Meja + SA was used to control the dwarf bunt pathogen in the roots and coleoptiles by activating  www.nature.com/scientificreports/ different root defence genes. SA is a defence signal molecule associated with resistance to hemibiotrophic and biotrophic pathogens 3,32,33 . Many defence-related genes of plants are associated with the SA pathway 18,34 . Our results showed that the expression of defence genes in roots and coleoptiles of SA-and Meja-treated plants was higher compared to control or non-treated plants (Figs. 1, 2, 3). Additionally, our results revealed that the invasion capability of T. controversa decreased with the passage of time after treatment with SA, Meja and Meja + SA (Fig. 4). Thus, we concluded that inducers of resistance significantly control TCK in the roots and coleoptiles. Movement of the hyphae into roots and into vegetative and reproductive parts of the plants almost disappeared after treatment for 3 weeks with inducers of resistance. Pathogenesis-related (PR) proteins play key roles in the defence mechanism of plants against various pathogen infections 35,36 . PR proteins are a group of functionally diverse inducible proteins that accumulate in plant tissue in response to pathogen infection 37 . PR proteins directly threaten pathogen integrity or release biochemicals by their enzymatic activity as elicitor molecules, which activates other plant defence-related pathways 38 . Most PR proteins play a role in fungal/oomycete pathogens. PR-10a and HRin1 are associated with the defence response in cereals, and they help plants to overcome the infection of pathogens 39,40 . PR-10a-like proteins have been described in the root epidermis and root hair cells of pea plants 41 . HRin1 activates the hypersensitive response in plants, which prevents the establishment of the pathogens 31 . Catalase genes are the major component of oxidative stress metabolism 27,28 . Catalases, along with other scavenging enzymes for reactive oxygen species (ROS), such as superoxide dismutase (SOD) and ascorbate peroxidase, are of fundamental importance to the survival of organisms undergoing oxidative and pathogenic stress 42 . Previous studies have shown that the expression of COI1-1 and COI1-2 genes enhances resistance to soilborne pathogens in various crops 30 . Meja signalling is mediated by COI1-1 and COI1-2 genes, which are F-box components of the Skp1-Cullin F-box protein (SCF COI1 ) ubiquitin E3 ligase 43 . COI is also involved in apical dominance 44 , leaf senescence 45 , ethylene-induced root growth inhibition in the light in Arabidopsis thaliana 46 and inositol polyphosphates 47 . Therefore, we applied the inducers of resistance to the highly susceptible wheat cultivar to activate the defence genes in the roots and coleoptiles to reduce the level of dwarf bunt pathogen. The roots and coleoptiles of wheat seedlings exhibited changes in gene expression during T. controversa inoculation and treatment with the inducers of resistance. The expression of defence-related gene expression of inoculated and treated plants was higher than the control plants (Figs. 1, 2, 3).
In this study, we also analysed the effect of T. controversa on anther development to investigate whether T. controversa present on epidermal and sub-epidermal cells of the anther and its intensity level increase on anther cells with the passage of time. TCK severity increased over time, resulting in cell wall rupturing in plants (Figs. 5, S1). Infection of young anthers prior to cell-fate differentiation results in total disruption of anther internal lobe development because all sub-epidermal cells are abnormal and cannot produce differentiated features of the EN, ML and PMCs 48 . Fungal penetration into roots is a gradual progress. Spores start to germinate on the soil surface, enter into roots, move to nodes and finally reach into anthers. We first observed fungal hyphae only on EPI cells, but over time, the fungal hyphae appeared on the EN, ML and PMCs (Fig. 5). These results indicated that establishment of hyphae on cells and sub-cells destroys the normal process of cell expansion and division. Additionally, the intensity level of the hyphae increased as the anther increased in size and matured (Fig. S1). Healthy anthers produced healthy ovaries and grains, while infected anthers produced bunted ovaries. Finally, ovaries and grains produced by the infected anthers turned into a black coloured powder containing teliospores (Figs. S2, S3, S4).
Our finding supported the hypothesis that inducers of resistance activate the defence responses in the roots and coleoptiles and that they eliminate the pathogen movement to the vegetative and reproductive parts of the plants. Exogenous application of inducers of resistance was effective in reducing the infection of T. controversa into roots, coleoptiles and anther tissues. Our results revealed that treatment with the inducers of resistance decreased the intensity level of the T. controversa hyphae at 3 weeks with no obvious systems observed at any stage of the plants and no teliospores in the grains. In contrast, clear hyphae were observed on the epidermis and sub-epidermal cells of the anthers in non-treated inoculated plants, and intensity level of hyphae increased with the passage of time as the anthers matured. Additionally, plant height was reduced by T. controversa but increased by the inducers of resistance. www.nature.com/scientificreports/  The Dongxuan 3 specimens (30-day seedlings) were grown in a greenhouse incubator (Percival, America) under a 24 h light (6 ± 2 °C and 70% relative humidity) regime. The wheat seedlings were inoculated with TCK according to the method of our laboratory. Briefly, TCK was grown on 2% agar media and incubated for 60 days at 5 °C under 24 h light. The TCK mycelia were harvested in a laminar flow hood by adding 5 ml of ddH 2 O into each TCK culture plate. TCK hyphae were injected 5 times into the root zone at 2-day intervals. Hyphae were also injected into spikes during ear emergence from boot, corresponding to Zadok's growth stages 49, 50 and 51 49 . The TCK-inoculated plants were kept in an incubator at 6 ± 2 °C until they were treated with the inducers of resistance.
The inducers of resistance were applied 6 and 8 days after TCK inoculation into the wheat root zones and seedlings as previously reported 17  RNA extraction and cDNA synthesis. The roots and coleoptiles were harvested, immediately frozen in liquid nitrogen and ground in a 2-µL centrifuge tube by adding 5-mm beads. Total RNA was extracted from 0.8 g of root and coleoptile powder using the Solarbio Life Sciences kit (Beijing, China) according to the manufacturer's instructions. The quantity and quality of RNA were analysed by a NanoDrop device (Denovix, Spectrophotometer, USA). First-strand cDNA was synthesized using 1 µg of purified total RNA, RT/RI enzyme and oligo (dT) 18 primer (TransGen) following the kit's instructions (TransGen). The synthesized cDNA was diluted 4 times in ddH 2 O to a final 80 µL volume, and 2 µL of the diluted cDNA was used as the template for real-time PCR analysis.
Real-time quantitative RT-PCR (RT-qPCR) analysis of defence-related genes. RT-qPCR was performed using Top Green qPCR SuperMix (TransGen) in a volume of 20 µL according to the manufacturer's instructions and applied to the QuantStudio 5 real-time PCR system (Applied Biosystems, Beijing, China) for quantification. Amplification of the wheat GPDH43 gene was used as an internal control for normalizing all data. We used the following RT-qPCR protocol: pre-denaturation at 95 °C for 10 min; and 40 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s. The 2 −ΔΔCt method 50 was used to evaluate the relative expression of defencerelated genes with three biological replicates and four technical replicates.
Staining TCK in root and coleoptile tissues. Hyphae in root and coleoptile segments were stained with the wheat germ agglutinin-Alexa Flour 488 conjugate (WGA-AF 488) chitin-specific dye (Molecular probes/ Invitrogen, P1304MP, Life Technologies, America), and plant cells were stained with propidium iodide (PI). Depending on the experiment, roots and coleoptiles were dipped in absolute ethanol for 30 min. Absolute ethanol was added three times after 35 min until the tissue turned white. Subsequently, both segments were incubated at room temperature for 5 min in 1 × phosphate-buffered saline (PBS; pH 7.4) containing each respective dye at 10 µg/ml. This process was repeated three times for optimal washing. PI (W11261, Life Technologies, USA) was used as a counterstain by adding it to the WGA-AF 488 staining solution at a final concentration of 10 µg/ml. Each segment of root and coleoptile was mounted on a glass slide for observation using a confocal microscope (TCS SP8, Leica, Germany). WGA-AF 488 was excited at a wavelength of 448 nm and detected at 510-550 nm.
PI was excited at a wavelength of 561 nm and detected at 570-730 nm. For every treatment, 15 to 20 replicates were assessed.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Impact of Germination and Fermentation on Rheological and Thermo-Mechanical Properties of Wheat and Triticale Flours

: Common cereal processing through germination and fermentation usually has an important impact on the technological performance of the ﬂours, mainly because of the activation of endogenous enzymes acting on macromolecules. The aim of the present study is to estimate the e ﬀ ect of germination and fermentation, using a mixture of Lactobacillus casei and Kluyveromyces marxianus subsp. marxianus , on the rheological properties of di ﬀ erent wheat and triticale varieties. Moreover, the thermo-mechanical behaviour of the white wheat ﬂour-based dough, including germinated grain ﬂour or sourdough was also tested. Grains germination and sourdough fermentation exerted a high inﬂuence on the rheological behaviour of the ﬂour-based suspensions. Germination a ﬀ ected the structure and stability of the suspensions, resulting in samples with viscous behaviour prevailing over the elastic one. The temperature ramp tests revealed that germination together with fermentation lead to higher resistance to temperature changes. In agreement with the results of the rheological investigations on rheometer, the Mixolab test performed on ﬂour obtained from germinated grains revealed lower dough stability and protein weakening at temperature increase. On the other hand, a signiﬁcant improvement of the pasting properties of the dough was obtained when adding sourdoughs to the wheat ﬂour. increased hydrolytic activity of the enzymes interfered with gel formation at thermal treatment. The rheological tests indicated that combining grains germination and sourdough fermentation results in increased gelatinization temperature domains. The addition of 15% ﬂour obtained from germinated wheat or triticale signiﬁcantly a ﬀ ected protein weakening, starch gelatinization, the stability of the starch gel at high temperature and starch retrogradation, causing signiﬁcant shortening of dough stability.

Introduction
Bread products obtained from refined wheat flour have relatively poor nutritional value, being low in mineral, vitamin and fibre content [1]. Recently, many studies focused on different processing techniques, aiming to enhance functional properties of wheat flour and quality of the final products. Besides fortification by addition of pseudocereals or leguminous flours to the wheat flour, fermentation and germination were proved to be simple techniques for improving the nutritive and functional properties of grains [2][3][4]. In particular, Sibian et al. [5] showed that germination improved functional properties of both wheat and triticale flours.
During germination, the main storage biopolymers of cereal grains, namely carbohydrates, proteins and lipids are hydrolysed to lower molecular weight compounds, because of the activity of the hydrolytic enzymes, which are inactive in raw seeds [6]. These processes result in improved digestibility, bioavailability and bioaccessibility of various nutrients and biologically active molecules. In addition, the use of sourdough in bread-making technology leads to increasing levels of numerous bioactives [3,4,[7][8][9]. In particular, the mineral bioavailability is improved through the activity of phytate degrading enzymes, which exist in yeast and lactic acid bacteria isolated from sourdough, whereas the digestibility of starch is reduced in the presence of lactic acid produced during sourdough fermentation, resulting in low glycemic index products [1,7].
Both cereal grains germination and sourdough fermentation were reported to significantly influence the technological performance of the flours and doughs. Many studies were focused on the effect of addition of germinated legumes or pseudocereals, such as pea [10], soy [11], broadbean [12], amaranth [6] etc., on the rheological properties of the wheat flour-based dough. Anyway, there are fewer studies regarding the effect of wheat germination and/or fermentation on dough rheology. Singh et al. [13] reported that wheat germination resulted in a softened dough structure, characterized by lower values of the elastic modulus and viscous modulus. A similar effect on the rheological properties caused by sourdough addition to the wheat flour was reported by Clarke et al. [14]. They showed that sourdough addition allowed obtaining more easily extensible doughs. On the other hand, Angioloni et al. [15] reported less elastic and firmer doughs, most probably as a result of physicochemical changes occurring in the protein network induced by the addition of sourdough. The addition of sourdough lowers the pH of the dough, affecting the structure and properties of different flour components, such as gluten and starch, which influences to a high extent the rheological behaviour of the dough [15].
The aim of the present study is to investigate the impact of the changes induced by wheat and triticale germination and sourdough addition on the rheological properties and thermomechanical behaviour of the dough. In this respect, sourdough samples were obtained through fermentation of the wholemeal flour suspensions using a mixture of lactic acid bacteria and yeast.

Materials
Three wheat cultivars bred in Romania, namely Gabriela (G) and Trivale (T), that are winter type common wheat (Triticum aestivum L.), and spelt (S) (Triticum aestivum subsp. spelta) and one triticale (X Triticosecale Wittmack) cultivar (TT) were used in the study.
The commercial white wheat flour (moisture content of 13.18%, protein content of 9.40%, and ash content of 0.48%) was purchased from the local market (Galati, Romania). All chemicals used in the experiment were of analytical grade.

Germination of Wheat and Triticale Grain
The grain samples used in the study were first washed with tap water and then sanitised using aqueous ethanol solution (70%). After a final rinsing with distilled water, the grains were evenly distributed on sterile cotton layers, moistened and allowed to germinate for four days at 23 • C ± 1. The germinated Gabriela (gG), Trivale (gT), spelt (gS) and triticale (gTT) samples further used in the experiment were obtained by drying at 55 • C ± 2 for 48 h in a convection oven (LabTech LDO-030E, Daihan LabTech Co., LTD, Kyonggi-Do, Korea).

Flour Obtaining
The raw and germinated grain samples were milled with a laboratory mill (WZ-2, Sadkiewicz Instruments, Bydgoszcz, Poland) into flours with particles size lower than 315 µm. The obtained flours were stored in glass containers at~4 • C for further tests. The flour samples obtained by milling the raw grains were used as control for the germination experiment.

Sourdoughs Preparation
Inoculum consisted of a mixture of Lactobacillus casei (Nutrish ® ), and yeast Kluyveromyces marxianus subsp. marxianus (LAF4) from Chr Hansen, Brasov, Romania. Producer recommendations were followed to obtain an inoculum size of 10 8 cfu/100 g sourdough. The fermentation was initiated by mixing the wholemeal flour obtained from raw and germinated grains with tap water and inoculum, such as to get a final dough yield (100 × mass of dough/mass of flour) of 300. The fermentation of the sourdoughs was carried out in large beakers covered with aluminium foil at 40 • C for 20 h, using a laboratory incubator (Pol-Eko Aparatura, WodzisławŚląski, Poland).
For each type of flour used in the fermentation experiment, a control sample consisting of a flour suspension of the same concentration as in case of sourdoughs preparations, was prepared without inoculum addition.

Rheological Properties
The rheological properties of the control suspensions prepared using flours from raw (G, T, S and TT) or germinated grains (gG, gT, gS and gTT) and of the corresponding sourdough samples (G_SD, T_SD, S_SD and TT_SD prepared out of G, T, S and TT flours, and gG_SD, gT_SD, gS_SD and gTT_SD prepared out of gG, gT, gS and gTT flours) were measured using a controlled-stress rheometer (AR2000ex, TA Instruments, Ltd, New Castle, DE, USA) equipped with a Peltier temperature control system, and a plate-plate geometry (diameter of 40 mm). The closing gap was set to 2 mm and a solvent trap was used to avoid moisture loss during rheological tests.
For each sample the linear viscoelastic region (LVR) yield point was first identified through running stress sweep tests under oscillatory flow in small amplitude conditions, by increasing the stress values from 0.01 to 100 Pa, at an oscillation frequency of 1 Hz. The obtained data were used to determine the stress domain where the structure of the sample remained unaffected, known as the LVR. The intersection of the storage and loss moduli (G and G"), corresponding to the minimal stress value required for the proper beginning of flow, was also estimated [17]. Further dynamic frequency sweep tests were performed within LVR, at a constant oscillatory stress of 0.1 Pa. The working temperature was set at 20 • C for the stress and frequency sweep tests.
In order to study the structure changes induced by heating in the suspensions and sourdoughs, temperature ramp tests were carried out in quasi-static conditions (frequency of 1Hz, oscillatory stress of 0.1 Pa) by increasing the temperature from 20 to 90 • C by 1.5 • C/min. The samples were further maintained at 90 • C for 10 min. The temperature-induced gelatinization of the studied samples was estimated using the procedure proposed by Ding et al. [18]. The gelatinization temperature range was determined by calculating the first derivative of the third order equation used to fit the G vs. temperature values, in the area where inflection was observed. Only equations with r 2 approaching unity were considered proper for estimating the gelatinization temperature domain.

Thermo-Mechanical Properties
In order to estimate the influence of germination and fermentation processes on the thermo-mechanical properties of the wheat flour-based doughs, the Mixolab Chopin (Tripette & Renaud Chopin, Villeneuve La Garenne, France) was used.
The impact of germination was determined by performing measurements on mixtures consisting of 15% wholemeal flour obtained by milling the investigated raw and germinated grains and 85% white wheat flour. The flours obtained from raw (G, T, S and TT) and germinated (gG, gT, gS and gTT) grains were mixed with the white wheat flour directly into the Mixolab tank.
In the second part of the experiment the white wheat flour (85%) was supplemented with sourdoughs, prepared with raw and germinated wheat and triticale flours, in such ratio to provide the same amount of wholemeal flour (15%) as in the case of the first part of the experiment, when the influence of grains germination was tested.
The standard Chopin+ protocol was employed for the empirical rheological measurements, and the typical Mixolab parameters were registered for estimating the properties of the main flour constituents during mixing and thermal treatment. The amount of water added to the mixtures in the Mixolab tank was decided such as to reach the standard torque value of 1.1 Nm (C1) at initial mixing. In case of the samples including sourdough, the amount of added water was accordingly reduced. The following torque values were used to gather information on the impact of germination and fermentation on the thermo-mechanical performance of main components of the flours: C2 (Nm) which is a measure of protein weakening while applying double constraints to the dough, consisting of mechanical work and temperature raise, C3 (Nm) which gives indication on starch gelatinization, C4 (Nm) related to the stability of the starch gel at high temperature and C5 (Nm) which provides evidence on starch retrogradation in the cooling stage. Moreover, the cocking stability (C34) was estimated as the difference between C3 and C4.

Statistical Analysis
Two independent germination and fermentation experiments were conducted and the measurements were performed at least in triplicate. The average values are reported together with standard deviations. Analysis of variance (ANOVA) was used to identify significant differences among results. The one-way ANOVA and Tukey's test with a 95% confidence interval were applied using the Minitab 18 software. Regression analysis was performed to identify any potential relationships between the studied properties of the flour samples.

Influence of Germination and Fermentation on the Rheological Properties of Wheat and Triticale Flours
Wheat and triticale samples were subjected to germination for four days and were then dried to 93.09-93.91% d.w. The germination process caused a slight increase in the protein content (from 15.04% to 15.75% in case of G, from 13.01% to 13.81% in case of T, from 12.52% to 13.80% in case of S and from 12.42% to 12.80% in case of TT), and had no significant influence on the ash content, which varied between 1.30% (for G) and 1.73% (for TT).
Rheological behaviour of flour suspensions and sourdoughs over the oscillatory stress sweep test is presented in Figure 1. Analysing the evolution of the viscoelastic moduli as a function of increasing stress, one can see that, regardless of the germination status, the control samples of all studied grain varieties presented the highest G and G" values. A strong solid-like behaviour was noticed for all control samples up to stress values in the 0.7-7.9 Pa range, depending on the flour type. In the LVR, delta values were lower than 24 • . Germination affected the structure and stability of the suspension, resulting in formulations having poor rheological quality, with viscous behaviour prevailing over the elastic one. When compared to the raw grains, flour suspensions obtained from germinated samples presented significantly lower values of G and G" moduli, with narrower but still present linear viscoelastic regions in case of G and TT (Figure 1a,d). For the other two germinated wheat varieties, flour suspensions entered directly into the transition phase toward flowing, with rapid decrease of both storage and loss moduli. The control samples yielded much higher stress values in comparison to flour suspensions obtained from the corresponding germinated grains ( Table 1). The maximum yield stress value was obtained in the case of G variety, which also presented the highest protein content (15.04%). Significant correlation was established between protein content and yield stress (r 2 of 0.9009) in case of non-germinated wheat. Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 13  The sourdoughs obtained from raw and germinated wheat and triticale samples displayed viscous behaviour (G'' > G') from the beginning of the test. Song and Zheng [19] presented the involvement of important molecules from flours, such as starch and gluten proteins, in defining the rheological behaviour of the dough. Among the main components of the flours, starch was shown to exhibit mainly non-linear viscoelastic response [20,21], while gluten was reported to be responsible for dough elasticity, being highly resistant to stress sweep [17,20]. Our results indicated that germination and fermentation interfered with gluten performance, most probably as a result of increased protease activity. The decrease of the molecular weight of proteins and peptides, as well as the potential discontinuities in the gluten network caused by the protease assisted cleavage of the peptide bonds affected dough resistance to the applied stress.  The sourdoughs obtained from raw and germinated wheat and triticale samples displayed viscous behaviour (G" > G ) from the beginning of the test. Song and Zheng [19] presented the involvement of important molecules from flours, such as starch and gluten proteins, in defining the rheological behaviour of the dough. Among the main components of the flours, starch was shown to exhibit mainly non-linear viscoelastic response [20,21], while gluten was reported to be responsible for dough elasticity, being highly resistant to stress sweep [17,20]. Our results indicated that germination and fermentation interfered with gluten performance, most probably as a result of increased protease activity. The decrease of the molecular weight of proteins and peptides, as well as the potential discontinuities in the gluten network caused by the protease assisted cleavage of the peptide bonds affected dough resistance to the applied stress.
The rheological behaviour of the studied flour suspensions and sourdoughs under temperature constraint is shown in Figure 2. Higher G values were registered for the control samples obtained from raw grain flours at low temperatures, suggesting that the changes occurring in the grains during germination and in the flour matrix during fermentation, affected the rheological stability of the suspensions and sourdoughs.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 13 The rheological behaviour of the studied flour suspensions and sourdoughs under temperature constraint is shown in Figure 2. Higher G' values were registered for the control samples obtained from raw grain flours at low temperatures, suggesting that the changes occurring in the grains during germination and in the flour matrix during fermentation, affected the rheological stability of the suspensions and sourdoughs. Regardless of the investigated samples, in the first part of the heating stage, a slight decreasing trend of G' was observed ( Figure 2). This phenomenon could be associated to the heat-induced weakening of the proteins, and to a lower extent to the starch swelling process which tends to compensate the decrease of G' values, caused by gradual proteins weakening. Gänzle et al. [22] stated that degradation of gluten protein structures in sourdoughs affects the viscoelastic properties of the final dough, which explains the lower G' values in case of sourdoughs. Except for spelt, at low temperature all sourdough samples had significant lower G' values compared to the corresponding suspensions. However, after completion of the gelling process, at 80-90 °C, no significant differences were observed between suspensions and sourdoughs. The curves showing the evolution of the elastic modulus (G') were mainly grouped as a function of germination. As a result of the germination process, lower G' values were obtained after achieving the gelling point ( Figure 2). This behaviour might be attributed to starch, which competes with proteins for binding the water molecules available in the system. Over the upper limit of protein denaturation temperature range, the starch swelling prevails, further grabbing all available water. Jane et al. [23] stated that the amylose-to-amylopectin ratio highly influences the starch functional properties. Germination was found to increase the amount of amylopectin within starch granules [23,24]. Moreover, the high amount of dextrose and maltose formed through starch hydrolysis during germination as a result of increased amylolytic activity will interfere with gel formation. In this respect, Chinma et al. [25] observed that germination determined the increase of the least gelling concentration of Cyperus esculentus flours from 12 to 20%. Regardless of the investigated samples, in the first part of the heating stage, a slight decreasing trend of G was observed (Figure 2). This phenomenon could be associated to the heat-induced weakening of the proteins, and to a lower extent to the starch swelling process which tends to compensate the decrease of G values, caused by gradual proteins weakening. Gänzle et al. [22] stated that degradation of gluten protein structures in sourdoughs affects the viscoelastic properties of the final dough, which explains the lower G values in case of sourdoughs. Except for spelt, at low temperature all sourdough samples had significant lower G values compared to the corresponding suspensions. However, after completion of the gelling process, at 80-90 • C, no significant differences were observed between suspensions and sourdoughs. The curves showing the evolution of the elastic modulus (G ) were mainly grouped as a function of germination. As a result of the germination process, lower G values were obtained after achieving the gelling point ( Figure 2). This behaviour might be attributed to starch, which competes with proteins for binding the water molecules available in the system. Over the upper limit of protein denaturation temperature range, the starch swelling prevails, further grabbing all available water. Jane et al. [23] stated that the amylose-to-amylopectin ratio highly influences the starch functional properties. Germination was found to increase the amount of amylopectin within starch granules [23,24]. Moreover, the high amount of dextrose and maltose formed through starch hydrolysis during germination as a result of increased amylolytic activity will interfere with gel formation. In this respect, Chinma et al. [25] observed that germination determined the increase of the least gelling concentration of Cyperus esculentus flours from 12 to 20%. Therefore, higher dosage of germinated flour is needed to achieve a dough consistency similar to the non-germinated wheat flour.
In order to study the temperature-induced gelatinization of the suspensions and sourdoughs, the experimental results in the inflexion area of G vs. temperature curves ( Figure 2) were fitted using a third-order equation, and the gelatinization temperature domains were estimated by calculating the first derivative of these equations. Analysing the results presented in Figure 3 one can observe that germination together with fermentation determined the increase of the gelatinization temperature domains. Our results comply with Frias et al. [24], who showed that germination altered lentil starch granule surface, leading to higher resistance to temperature changes.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 13 Therefore, higher dosage of germinated flour is needed to achieve a dough consistency similar to the non-germinated wheat flour. In order to study the temperature-induced gelatinization of the suspensions and sourdoughs, the experimental results in the inflexion area of G' vs. temperature curves ( Figure 2) were fitted using a third-order equation, and the gelatinization temperature domains were estimated by calculating the first derivative of these equations. Analysing the results presented in Figure 3 one can observe that germination together with fermentation determined the increase of the gelatinization temperature domains. Our results comply with Frias et al. [24], who showed that germination altered lentil starch granule surface, leading to higher resistance to temperature changes. Temperature domains corresponding to the gelling process for the studied suspensions and sourdoughs during the temperature ramp test. The analysed temperature ranges corresponding to the inflection area of G' vs. t curves are represented with lines, whereas the estimated gelling temperature domains are indicated as columns. Tests were performed on suspensions prepared using flours from raw (G, T, S and TT) or germinated grains (gG, gT, gS and gTT) and on corresponding sourdough samples (G_SD, T_SD, S_SD and TT_SD prepared out of G, T, S and TT flours, and gG_SD, gT_SD, gS_SD and gTT_SD prepared out of gG, gT, gS and gTT flours).

Influence of Germination and Fermentation on the Thermo-mechanical Properties of Doughs
The impact of grains germination and sourdough fermentation on the bread making properties of the white wheat flour, was estimated by monitoring the thermo-mechanical behaviour of the doughs by means of Mixolab device. The main Mixolab parameters registered when supplementing the white wheat flour with 15% wholemeal flours from raw or grains or corresponding sourdoughs are presented in Table 2.

Influence of Grain Germination
The addition of 15% wholemeal flour obtained through grinding the germinated G, T, S and TT grain samples to the white wheat flour resulted in significant shortening of dough stability (DS) compared to the corresponding raw samples. The DS values ranged from 8.82 to 9.93 min in case of the doughs prepared with raw grain flour, and from 2.68 to 4.03 min for the samples with germinated grain flour (Table 2). Our results are in agreement with Rosales-Juarez et al. [11] who tested the influence of germinated and non-germinated soybean flour on the rheological behaviour of the doughs and, regardless of the addition level, reported shorter stability of the wheat flour-based dough samples supplemented with germinated soy flour. On the other hand, Sadowska et al. [10] reported dough stability prolongation for the wheat flour-based samples, including different

Influence of Germination and Fermentation on the Thermo-Mechanical Properties of Doughs
The impact of grains germination and sourdough fermentation on the bread making properties of the white wheat flour, was estimated by monitoring the thermo-mechanical behaviour of the doughs by means of Mixolab device. The main Mixolab parameters registered when supplementing the white wheat flour with 15% wholemeal flours from raw or grains or corresponding sourdoughs are presented in Table 2.

Influence of Grain Germination
The addition of 15% wholemeal flour obtained through grinding the germinated G, T, S and TT grain samples to the white wheat flour resulted in significant shortening of dough stability (DS) compared to the corresponding raw samples. The DS values ranged from 8.82 to 9.93 min in case of the doughs prepared with raw grain flour, and from 2.68 to 4.03 min for the samples with germinated grain flour ( Table 2). Our results are in agreement with Rosales-Juarez et al. [11] who tested the influence of germinated and non-germinated soybean flour on the rheological behaviour of the doughs and, regardless of the addition level, reported shorter stability of the wheat flour-based dough samples supplemented with germinated soy flour. On the other hand, Sadowska et al. [10] reported dough Appl. Sci. 2020, 10, 7635 8 of 12 stability prolongation for the wheat flour-based samples, including different percentages of germinated pea flour, compared to the samples with raw pea flour. When the dough was subjected to the dual constraint of mechanical shear and temperature, the consistency decreased to a minimum torque (C2) varying with the flour mixture and type of processing (Table 2). Higher C2 values were registered for dough samples with raw grain flour addition (0.42-0.47 Nm), compared to the samples prepared with corresponding germinated grain flour (0.12-0.17 Nm). The decrease of both C2 and DS values observed when testing flour from germinated grains, can be explained by the advanced disruption of the protein network caused by peptidase formed during germination. The protein aggregates stabilised through the disulfide or dityrosine linking and hydrogen bonds are essentially responsible for dough consistency and stability [26]. According to Bigiarini et al. [27], gliadins are degraded during germination into small peptides, the enzymes mainly responsible for this activity being cysteine proteinase and carboxypeptidases, which are highly active during wheat germination. Table 2. Effect of the addition of flours from raw (G, T, S and TT) or germinated grains (gG, gT, gS and gTT) and of corresponding sourdough samples (G_SD, T_SD, S_SD and TT_SD prepared out of G, T, S and TT flours, and gG_SD, gT_SD, gS_SD and gTT_SD prepared out of gG, gT, gS and gTT flours). On the thermo-mechanical properties of the white wheat flour (WF) dough. Measurements were performed in triplicate by means of Mixolab using the Chopin+ protocol. As shown in Table 2 and Figure 4, the use of gG, gT, gS and gTT flours resulted in changes of the pasting properties compared to the corresponding dough samples with raw G, T, S and TT flour (controls). It should be noted that addition of raw TT and S flour had a higher influence on pasting properties compared to G and T. The highest values of the maximum torque during heating (C3) were 1.99 and 2.20 Nm in case of the samples with raw G and T flour addition. Weaker starch gel stability (C4) was registered for dough samples with S and TT, most probably because of the high amylase activity in these samples, compared to G and T. Triticale and spelt tend to have higher α-amylase activity compared to wheat, depending on the cropping environment [28,29].

Samples
Different tendency of the Mixolab curves was registered when germinated grain flours were added to the white flour to obtain dough samples (Figure 4b). Germination induced the increase of amylase activity and consequently starch hydrolysis became very evident after the gelatinization. Therefore, significantly lower C4 values were registered for the dough samples including germinated grain flours. The cooking stability (C34) ranged from 0.3 Nm to 0.68 Nm, suggesting that the amylase activity continued even during the Chopin+ stage when the dough is heated at 90 • C. According to Dubat and Boinot [30], the (C34) values increased from 0.11 Nm to 0.46 Nm when adding exogenous maltogenic amylases to the wheat flour. Similar reduction was observed for the torque values, which gives indication about starch retrogradation (C5). When studying the influence of brown rice and soy germination on the bread-making performance of the flours, Cornejo and Rosell [31] and Patraşcu et al. [32] registered lower torque values while heating the dough up to 90 • C and at further cooling down to 50 • C. They explained this change in the thermo-mechanical behaviour of the dough through the extensive hydrolysis of the starch by the amylases-activated during germination.  Different tendency of the Mixolab curves was registered when germinated grain flours were added to the white flour to obtain dough samples (Figure 4b). Germination induced the increase of amylase activity and consequently starch hydrolysis became very evident after the gelatinization. Therefore, significantly lower C4 values were registered for the dough samples including germinated grain flours. The cooking stability (C34) ranged from 0.3 Nm to 0.68 Nm, suggesting that the amylase activity continued even during the Chopin+ stage when the dough is heated at 90 °C. According to Dubat and Boinot [30], the (C34) values increased from 0.11 Nm to 0.46 Nm when adding exogenous maltogenic amylases to the wheat flour. Similar reduction was observed for the torque values, which gives indication about starch retrogradation (C5). When studying the influence of brown rice and soy germination on the bread-making performance of the flours, Cornejo and Rosell [31] and Patraşcu et al. [32] registered lower torque values while heating the dough up to 90 °C and at further cooling down to 50 °C. They explained this change in the thermo-mechanical behaviour of the dough through the extensive hydrolysis of the starch by the amylases-activated during germination.

Influence of Sourdough Addition
Regardless of the investigated grain sample, the addition of sourdoughs prepared with raw grain flours (G_SD, T_SD, S_SD and TT_SD) resulted in doughs with lower DS and C2 values (Table

Influence of Sourdough Addition
Regardless of the investigated grain sample, the addition of sourdoughs prepared with raw grain flours (G_SD, T_SD, S_SD and TT_SD) resulted in doughs with lower DS and C2 values ( Table 2). The sourdough fermentation caused the DS shortening from 8.82-9.93 min to 5.23-6.32 min, and the C2 diminishing from 0.42-0.47 Nm to 0.31-0.37 Nm. On the other hand, the addition of sourdough prepared with germinated grain flour resulted in higher DS and lower C2 values compared to the corresponding samples with raw grain flour. This trend registered for the dough samples including sourdoughs might be the result of the low pH value. In agreement with Bottari et al. [33], the optimal pH for gliadin hydrolysis is 4.25. Moreover, Banu and Aprodu [26] and Clarke et al. [34] showed that dough behaviour during mixing at 30 • C and during temperature increase from 30 to 55-60 • C is influenced by pH, affecting both DS and C2 values. Similar results for DS and C2 were obtained when preparing dough with raw and germinated soybean flour added to the wheat flour [32]. The authors explained the reduction of C2 values by protein weakening during dough mixing and heating by 4 • C/min, but also by the action of proteolytic enzymes that are activated during germination.
The dough samples prepared with sourdoughs made of raw G, S and T flours displayed similar pasting properties, the torque values being significantly higher with respect to the TT-based sample ( Figure 5). For instance, the values of C5 that defines starch retrogradation, ranged from 2.25 Nm, corresponding to the sample with TT-based sourdough, to 2.72 Nm in case of the sample with S, suggesting different optimal end-use for each sample. According to Dubat and Boinot [30] the flours suitable for bread making, brioche and biscuits have C5 of 2.05, 2.43 and 2.75 Nm, respectively. pH for gliadin hydrolysis is 4.25. Moreover, Banu and Aprodu [26] and Clarke et al. [34] showed that dough behaviour during mixing at 30 °C and during temperature increase from 30 to 55-60 °C is influenced by pH, affecting both DS and C2 values. Similar results for DS and C2 were obtained when preparing dough with raw and germinated soybean flour added to the wheat flour [32]. The authors explained the reduction of C2 values by protein weakening during dough mixing and heating by 4 °C/min, but also by the action of proteolytic enzymes that are activated during germination.
The dough samples prepared with sourdoughs made of raw G, S and T flours displayed similar pasting properties, the torque values being significantly higher with respect to the TT-based sample ( Figure 5). For instance, the values of C5 that defines starch retrogradation, ranged from 2.25 Nm, corresponding to the sample with TT-based sourdough, to 2.72 Nm in case of the sample with S, suggesting different optimal end-use for each sample. According to Dubat and Boinot [30] the flours suitable for bread making, brioche and biscuits have C5 of 2.05, 2.43 and 2.75 Nm, respectively. Grains germination improved starch gelatinization, starch stability and starch retrogradation properties of the dough samples prepared with sourdough ( Table 2). The decrease of the pH during sourdough fermentation limited the activity of α-amylase, and consequently improved starch stability. The most significant improvement of the pasting properties was observed in case of the dough sample including sourdough prepared with germinated G flour. Grains germination improved starch gelatinization, starch stability and starch retrogradation properties of the dough samples prepared with sourdough ( Table 2). The decrease of the pH during sourdough fermentation limited the activity of α-amylase, and consequently improved starch stability. The most significant improvement of the pasting properties was observed in case of the dough sample including sourdough prepared with germinated G flour.

Conclusions
Processing wheat and triticale through germination and further sourdough fermentation affected the rheological and thermo-mechanical properties of the flours. Regardless of the investigated grain, a narrower viscoelastic domain was obtained for samples subjected to germination and/or fermentation. The partial hydrolysis of the starch and proteins during germination as a result of increased hydrolytic activity of the enzymes interfered with gel formation at thermal treatment. The rheological tests indicated that combining grains germination and sourdough fermentation results in increased gelatinization temperature domains. The addition of 15% flour obtained from germinated wheat or triticale significantly affected protein weakening, starch gelatinization, the stability of the starch gel at high temperature and starch retrogradation, causing significant shortening of dough stability.