Role of genetic diversity and salicylic acid in drought stress memory of tall fescue

AbstractUnderstanding the concequence of drought stress memory and its interaction with genetic diversity and pollination system is critical for improving resilience in turf and forage grasses like tall fescue. With global warming and the predicted occurrence of frequent drought stresses in the future, little is known about these effects, especially on morphological traits, physiological responses, root characteristics, and spectral reflectance indices (SRIs). This study addresses the knowledge gap by evaluating the effects of drought memory in tall fescue using four parental clones (two drought-sensitive and two drought-tolerant), which were manually controlled to produce four selfed (S1) and four open-pollinated (OP) genotypes. Over two years, these genotypes were exposed to five moisture treatments: control (C), two treatments with twice applications of drought stress (primary mild drought stress in two different stages and secondary at the end stage, D1t1D2 and D1t2D2), one severe drought stress treatment (secondary only, D2), and foliar spray of salicylic acid (SA) under end-stage drought stress (H2D2). Selfing induced inbreeding depression, reducing relative water content (RWC), growth, chlorophyll, carotenoid content, catalase activity and root length. Alterations in natural plant mating systems can modify the genetic structure of tall fescue germplasm. Drought memory (D1t1D2 and D1t2D2) improved RWC, root-to-shoot ratio, and most physiological traits, especially pigment content, particularly in drought-tolerant and OP genotypes. SA treatment was more effective in mitigating drought effects in S1 than OP. Significant genetic variation in SRIs was observed, indicating their potential as predictive tools physiological traits. These findings provide insights into breeding strategies and highlight the importance of leveraging drought memory and genetic diversity to enhance drought resilience in tall fescue.

IntroductionTall fescue (Festuca arundinacea Schreb., syn. Lolium arundinaceum [Schreb.] Darbysh.) is an outcrossing allohexaploid (2n = 6x = 42) turf and forage grass with substantial agricultural and ecological significance1. Like many grass species, tall fescue has evolved as an open-pollinated species due to its self-incompatibility mechanism. Therfore, inbreeding depression is a significant concern in this cross-pollinated species due to their reliance on genetic diversity for fitness and adaptability. This characteristic naturally drives breeding efforts toward creating superior synthetic cultivars and maintaining genetically diverse populations through methods such as polycross and open pollination among various genotypes2. Breeders utilize obligate cross-pollination in these plants to conduct genetic studies3. Understanding the genetic mechanisms associated with the reproductive system is essential for optimizing breeding strategies and enhancing important traits, such as drought stress tolerance, in species like tall fescue. Additionally tall fescue demonstrates notable potential for dehydration avoidance under water-deficit conditions compared to other cool-season grasses, largely due to its extensive rooting system, physiological traits such as capacity for osmotic adjustment under stress1.Drought stress is one of the most significant abiotic stressors affecting plant growth, productivity, and survival worldwide. It disrupts essential physiological processes, including plant water status, CO2 assimilation, oxidative balance, root development, and enzymatic activities, ultimately reducing biomass accumulation and photosynthetic efficiency4. Given the importance of these traits in determining drought resilience, their comprehensive assessment under stress conditions is critical1. To mitigate the adverse effects of drought, various strategies have been explored, including the exogenous application of plant growth regulators. Among these, salicylic acid (SA) has emerged as a promising candidate due to its multifaceted role in plant stress responses. Its an endogenous signaling molecule, plays a vital role in plant growth, flowering, defense, and stress responses5. Exogenous application of SA has been shown to improve drought tolerance and yield in tall fescue, underscoring its potential as a practical intervention to enhance stress resilience5,6.In addition to physiological and biochemical responses, plants exhibit stress memory, a phenomenon that enables them to respond more effectively to recurring stressors. Mild or short-term drought stress can trigger memory responses such as imprinting, priming, hardening, and acclimation, leading to enhanced stress tolerance7. Extensive research has demonstrated that prior exposure to drought influences future plant responses by upregulating specific stress memory genes8,9. These genes include dehydration-responsive element-binding (DREB) transcription factors, responsive to desiccation (RD) genes, and 9-cis-epoxycarotenoid dioxygenase (NCED) genes, which regulate stress adaptation. Their activation enhances the transcription of stress-responsive proteins, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), late embryogenesis abundant (LEA) proteins, and abscisic acid (ABA)-related enzymes, which collectively mitigate oxidative stress, maintain cellular homeostasis, and improve drought resilience10.In addition to genetic mechanisms, epigenetic modifications, heritable changes in gene expression without alterations in the DNA sequence, play a crucial role in drought stress memory. These modifications include DNA methylation, histone modifications, and small RNA-mediated regulation, which influence gene expression patterns in response to environmental stimuli11. For example, Arabidopsis plants subjected to recurrent dehydration cycles exhibit physiological and transcriptional memory, such as partially closed stomata during recovery periods, enhancing water conservation12. Similarly, Arrhenatherum elatius plants pre-exposed to drought exhibit greater biomass accumulation under subsequent drought stress compared to those experiencing drought for the first time9. Furthermore, increased genetic diversity among parental genotypes has been linked to enhanced drought stress memory in Dactylis glomerata progenies13. Despite these findings, limited research has been conducted on the role of drought memory and and its potential to enhance stress resilience in tall fescue.Traditional methods for evaluating drought tolerance typically rely on morphological, physiological, biochemical, and molecular assessments. However, these approaches are often time-consuming and involve destructive sampling. Visible-near infrared (Vis-NIR) spectroscopy offers a rapid, non-destructive alternative for assessing plant physiological status. This technique analyzes the interaction between electromagnetic radiation and plant tissues to estimate their chemical and biological composition14,15. Visible spectroscopy (400–750 nm) provides insights into plant pigments such as chlorophyll, anthocyanins, and carotenes, whereas near-infrared spectroscopy (NIR) primarily measures macro-components, particularly water content16,17. Various spectral indices derived from reflectance spectra, including the simple ratio (SR), normalized difference vegetation index (NDVI), stress index (SI), and water index (WI), have been developed to assess plant stress and physiological status18. These spectral indices provide valuable insights into plant responses to drought stress, enabling faster and more precise evaluations of drought tolerance in tall fescue. For instance, NDVI and SR have been correlated with biomass accumulation19, while indices such as mSR750/705 and mND750/705 effectively distinguish salinity stress18. Additionally, WI and normalized difference water index (NDWI) serve as sensitive indicators of plant water status, with WI demonstrating strong correlations with drought tolerance parameters20,21.Over the past decade, substantial progress has been made in drought tolerance research at Isfahan University of Technology, where superior tall fescue genotypes have been identified1,5,22,23. However, significant knowledge gaps remain regarding the effects of recurrent drought stresses, the exogenous application of SA, inbreeding depression (relative to open pollination), and their interactions on morphological, physiological, root traits and spectral reflectance indices. To address these gaps, the present study aims to: (1) investigate the impact of drought memory and SA application compared to single drought stress on morphological, physiological, root traits, and spectral reflectance indices; (2) evaluate the responses of selfed (S1) and open-pollination (OP) genotypes derived from drought-sensitive and drought-tolerant parents; and (3) determine correlations between these characteristics and spectral reflectance indices to develop preliminary models for indirect selection of drought-resilient genotypes.Results Analysis of variance and mean comparisonsAnalysis of variationResults from the combined analysis of variance (ANOVA) for 2019 and 2020 revealed significant effect (P < 0.05) of genotypes (G), pollination systems (P), and moisture treatments (T) on most measured traits, including morphology, physiology and root characteristics (Supplementary Tables S2-S4) and indices (not shown). Significant interactions were observed for G×P, G×T, and P×T, as well as the triple interaction of G×P×T, indicating that genotype, pollination system, and moisture treatment collectively influenced traits variation (Supplementary Tables S2-S4).Effects of pollination systems on traits and indicesMean comparison for pollination systems showed that open-pollinated (OP) plants exhibited increased crown diameter (CRD) by 15.22%, plant height (PH) by 21.44%, relative water content (RWC) by 15.09%, plant height in recovery (PHR) by 12.13%, chlorophyll a (Chla) by 6.36%, carotenoid content (Car) by 8.62%, total chlorophyll (Tchl) by 4.33%, catalase (CAT) activity by 8.01%, root length (RL) by 23.95% and the root-to-shoot ratio in recovery (R/SR) by 40.67% compared to selfed (S1) plants (Tables 1, 2 and 3). Conversely, S1 plants had higher wet forage yield (WFY) by 28.91%, dry forage yield (DFY) by 32.65%, the Chl a/b ratio by 2.08%, TChl/Car by 5.11%, ascorbate peroxidase (APX) activity by 7.31%, peroxidase (POX) activity by 16.76%, and most root characteristics, with increases of over 40% (Tables 1, 2 and 3). Emergence rates were lower in selfed genotypes, indicating inbreeding depression, although substantial variation was observed within populations (Fig. S2).Table 1 Mean comparison of tall fescue genotypes, two different pollination systems ((selfed (S1) and open-pollinated (OP)) and five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) for morphological traits and relative water content during the vegetative stage in two years (2019 and 2020).Full size tableTable 2 Mean comparison of tall fescue genotypes, two different pollination systems ((selfed (S1) and open-pollinated (OP)) and five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) for physiological traits measured in leaf tissue during the vegetative stage in two years (2019 and 2020).Full size tableTable 3 Mean comparison of tall fescue genotypes, two different pollination systems ((selfed (S1) and open-pollinated (OP)) and five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) for root traits during the vegetative stage in two years (2019 and 2020).Full size tablePearson’s correlation analysis was used to assess spectral reflectance indices, leading to the removal of five highly correlated indices: pigment specific normalized different (PSND), structure intensive pigment index (SIPI), red normalized difference vegetation index (RNDVI), photochemical reflectance index (PRI), and plant senescence reflectance index (PSRI) (Table S5). Mean comparison showed that OP genotypes exhibited higher values for simple ratio (SR), ratio analysis of reflectance spectra (RARSb), pigment specific simple ratio (PSSR), carotenoid reflectance index (CRI), and anthocyanin reflectance index (ARI), but lower values for ratio analysis of reflectance spectra (RARSa) and red/green ratio (RGR) compared to S1 genotypes (Table 4).Table 4 Mean comparison of tall fescue genotypes, two different pollination systems (selfed (S1) and open-pollinated (OP)) and five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) for spectral reflectance indices during the vegetative stage in two years (2019 and 2020). Mean followed by the same letter is not significantly different according to LSD test (probability level of 5%).Full size tableDrought stress effects on traits and indicesSevere drought stress (D2) significantly reduced PH, RWC, forage yield (WFY and DFY), chlorophyll pigments (Chla and Chlb), Car, Tchl, Tchl/Car, and CAT and POX activities. This reduction was 23% for dry forage yield. However, Pro, APX activity, and all root traits exhibited an increase under drought conditions (Tables 1, 2 and 3). Moreover, severe drought stress reduced most spectral indices, with the exception of RARSb, PSSR, green normalized difference vegetation index (GNDVI), normalized difference red edge index (NDRE), ARI, green difference vegetation index (GDVI) and RGR (Table 4). Pre-exposure effects on traits and indicesPre-exposure to drought (D1t1D2 and D1t2D2) improved RWC, chlorophyll pigments, Car, Tchl, and the root-to-shoot ratio (R/S and R/SR) compared to D2 (Tables 1, 2 and 3), providing evidence for drought memory. Additional enhancements were observed in Chl a/b, Tchl/Car, CAT, POX activities, and RL under D1t1D2, while PH, Pro, and APX activity were increased under D1t2D2 (Tables 1, 2 and 3). These results indicate that drought memory was more pronounced for physiological traits, particularly in water retention capacity, enzymatic activities, and root-shoot balance. RWC and Tchl decreased after the first drought event (D1), while Pro increased (Fig. S3a-f). Under OP and S1 populations, D1t1D2 and D1t2D2 treatments increased RWC compared to D2, further supporting the existence of drought memory (Fig. 1a). Spectral indices, including WI, NWI, RARSb, ARI, GDVI, and RGR, were enhanced under D1t1D2, while NDVI, RARSa, GNDVI, CRI, GDVI, and RGR improved under D1t2D2 (Table 4). Effects of salicylic acid (SA) application on traits and indicesFoliar SA application significantly improved physiological traits, RWC, PHR, root dry weight (RDW), R/S and R/SR under drought stress (Tables 1, 2 and 3). SA application during secondary drought stress (H2D2) notably increased RWC in OP genotypes (Fig. 1a). RWC trends over time indicated the highest values in C, D1t2D2, D1t1D2 and H2D2 treatments, while D2 had the lowest (Fig. 1b). Spectral indices such as SR, WI, NWI, RARSa, RARSb, and CRI improved under SA application in drought-stressed plants (Table 4).Fig. 1(A) Mean comparison of relative water content for the interactions of two different pollination systems (selfed (S1) and open-pollinated (OP)) and five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) during the vegetative stage in two years (2019 and 2020). Mean followed by the same letter is not significantly different according to LSD test (probability level of 5%). (B) The trend of relative water content for five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) based on the number of days (establishment) during two years (2019 and 2020).Full size imageInteraction effects Interaction effects of moisture treatments and pollination systems on traits and indicesOP populations under drought memory (D1t1D2 and D1t2D2) showed increased RWC, Car, Tchl, and R/S, while Pro, APX, and POX activities were higher under D1t2D2, and CAT activity and R/SR increased under D1t1D2. In contrast, S1 populations exhibited improvements only in Car, Tchl, and R/SR, suggesting drought memory was more pronounced in OP populations (Table 5). Under SA treatment (H2D2), OP plants had higher RWC, Tchl, Pro, POX activity, and R/S, whereas S1 plants showed increased PHR, Car, Tchl, CAT, POX activities, RDW, R/S, and R/SR, indicating SA was more effective in S1 plants (Table 5).Table 5 Mean comparison of interactions between moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) and two different pollination systems ((selfed (S1) and open-pollinated (OP)) for some morphological, physiological and root traits during the vegetative stage in two years (2019 and 2020). Mean followed by the same letter is not significantly different according to LSD test (probability level of 5%).Full size tableThe interaction effects of drought memory on spectral reflectance indices showed a significant increase in WI and RARSb in OP genotypes, while WI, ARI, GDVI, and RGR were enhanced in S1 genotypes under the D1t1D2 treatment compared to D2 (Table 6). Similarly, under the D1t2D2 treatment, NDVI, RARSa, CRI, and RGR increased in OP, whereas in S1, NDVI, RARSa, PSSR, GNDVI, NDRE, CRI, GDVI, and RGR showed significant improvements. The foliar application of SA (H2D2) at a concentration of 1 mM significantly enhanced WI, RARSa, and CRI in both OP and S1 genotypes under severe drought stress. However, SR, RARSb, PSSR, and GNDVI were improved only in S1 population (Table 6). Overall, drought stress memory and the application of salicylic acid positively influenced a greater number of spectral reflectance indices.Table 6 Mean comparison of interactions between moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) and two different pollination systems (selfed (S1) and open-pollinated (OP)) for some spectral reflectance indices during the vegetative stage in two years (2019 and 2020). Mean followed by the same letter is not significantly different according to LSD test (probability level of 5%).Full size table Interaction effects of moisture treatments and genetic diversity on traits and indicesDrought-sensitive genotypes under D1t1D2 and D1t2D2 exhibited greater RWC and Tchl improvements, while drought-tolerant genotypes showed higher Car, Tchl, CAT, POX activities, and R/SR, suggesting drought memory was stronger in tolerant genotypes (Table 7). SA application improved RWC, PHR, POX, RDW, R/S, and R/SR in sensitive genotypes, while tolerant genotypes benefited in Tchl, Pro, CAT, POX, R/S, and R/SR (Table 7).Table 7 Mean comparison of moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) separately for drought-sensitive and tolerant genotypes for some morphological, physiological and root traits during the vegetative stage in two years (2019 and 2020). Mean followed by the same letter is not significantly different according to LSD test (probability level of 5%).Full size tableThe response of spectral reflectance indices indicated that WI, RARSb, ARI, GDVI, and RGR significantly increased in drought-sensitive genotypes under D1t1D2 treatment compared to D2, while WI and RGR showed similar improvements in drought-tolerant genotypes (Table 8). Under D1t2D2 treatment, NDVI, RARSa, GNDVI, NDRE, CRI, and GDVI were enhanced in drought-sensitive genotypes, whereas NDVI, RARSa, PSSR, GNDVI, CRI, and RGR increased in drought-tolerant genotypes. The application of SA improved WI, RARSa and CRI in drought-sensitive genotypes under severe drought stress, while in drought-tolerant genotypes, it enhanced SR, RARSa, RARSb, and CRI (Table 8). These findings suggest that drought memory had a stronger effect on spectral reflectance indices in drought-sensitive genotypes. However, the foliar application of SA effectively mitigated drought stress in both drought-sensitive and drought-tolerant genotypes, as evidenced by improvements in various traits and indices.Table 8 Mean comparison of moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) separately for drought-sensitive and tolerant genotypes for some spectral reflectance indices during the vegetative stage in two years (2019 and 2020). Mean followed by the same letter is not significantly different according LSD test (probability level of 5%).Full size tablePrincipal component analysis (PCA) and genotype selectionPrincipal component analysis (PCA) was used to reduce data dimensionality and explore relationships between traits and genotypes. The first two principal components (PC1 and PC2) explained 52%, 54%, 63%, 56%, and 59% of the variation under the control (C), D1t1D2, D1t2D2, D2, and H2D2 treatments, respectively (Table S6).In the control treatment, PC1 showed strong positive correlations with DFYR, CRD, RV(A), RV(G), RA, RCL, and RDW, all of which are indicative of root production capacity and yield potential. Therefore, PC1 was designated as the ‘‘Root Characteristic System’’. PC2 exhibited negative correlations with Chla, Chlb, Car, Tchl, and Tchl/Car but a positive correlation with Chl a/b (Table S6). Since lower values for these traits indicate higher photosynthetic capacity, PC2 was labeled ‘‘Photosynthetic Capacity’’. The biplot analysis identified genotypes 11MS1 and 3MS1 as having strong root development and yield potential (Fig. 2).Fig. 2The biplot display of morphological, physiological, root traits and tall fescue genotypes during the vegetative stage in five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2). (PH: plant height, DFY: dry forage yield, PHR: plant height in recovery, DFYR: dry forage yield in recovery, RWC: relative water content, Chla: chlorophyll a content, Chlb: chlorophyll b content, Car: carotenoid content, Tchl: total chlorophyll, Chla/b: ratio of Chla/Chlb, Tchl/Car: ratio of Tchl/Car, Pro: proline content, CAT: catalase activity, APX: ascorbate peroxidase activity, POX: peroxidase activity, CRD: crown diameter, RL: root length, RV(A): root volume (archimedes), RV(G): root volume (giaroot), RA: root area, RCL: root cumulative length, RDW: root dry weight, R/S: root to shoot ratio, R/SR: root-to-shoot ratio in recovery).Full size imageUnder the D1t1D2 treatment, PC1 correlated positively with Chla, Car, Tchl, Chl a/b, R/S, and R/SR, suggesting that genotypes with higher PC1 values had enhanced photosynthetic capacity and were more stress-tolerant. In contrast, PC2 showed positive correlations with DFY, RWC, CRD, RV(A), RV(G), RA, RCL, and RDW (Table S6), indicating that genotypes with higher PC2 values were better suited for drought stress conditions. Biplot analysis highlighted genotypes 11MOP and 11MS1 as having high PC1 and PC2 values, marking them favorable candidates for initial drought exposure (Fig. 2).For the D1t2D2 treatment, PC1 exhibited negative correlations with DFYR, Chla, Tchl, RV(G), RA, RCL, and RDW, indicating that genotypes with high PC1 values had lower root production potential. Meanwhile, PC2 showed negative correlations with R/S, R/SR, CAT, and APX activities but a positive correlation with PHR (Table S6). Genotypes with lower PC2 values were considered more suitable for drought conditions. Based on the biplot analysis, genotypes 11MS1 and 3MS1, characterized by low PC1 and PC2 values, were identified as the most adapted to these conditions (Fig. 2).Under the D2 treatment, all open-pollinated (OP) genotypes clustered in regions associated with high PC2 values, which correlated with Tchl, Car, POX, CRD, R/SR, and RL (Fig. 2). Genotype 11MS1, with a combination of low PC1 and high PC2 values, was identified as a promising genotype for severe drought stress conditions.Under the H2D2 treatment, PC1 was strongly associated with DFY, DFYR, RV(A), RV(G), RA, RCL, and RDW, while PC2 showed positive correlations with Chla, Chlb, and Tchl (Table S6). PCA-based genotype classification indicated that 11MS1 had high potential for root and yield production (high PC1) and enhanced photosynthetic capacity (high PC2) under foliar SA application combined with drought stress (Fig. 2).Correlation among indices and traits; regression modelsThe correlation analysis between spectral reflectance indices and various traits in tall fescue genotypes (Table 9) revealed several significant relationships. The SR index showed positive correlations with CRD and PHR but negative correlations with Car and Pro. Similarly, WI was positively correlated with Chla, Chlb, Tchl, Tchl/Car, RL, and R/SR, while NWI exhibited positive correlations with PHR and Tch/Car but a negative correlation with R/S.Table 9 Correlation coefficients between spectral reflectance indices with different traits of tall fescue genotypes during the vegetative stage based on the average of two years (2019 and 2020) for eight genotypes (1MOP, 1MS1, 3MOP, 3MPS1, 11MOP, 11MS1, 21MOP and 21MS1), five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) and two replications (n = 80). * and ** show significance at the 0.05 and 0.01 probability levels, respectively.Full size tableRARSa was negatively correlated with PHR and RWC but positively correlated with Car, Pro, and R/S. Meanwhile, RARSb showed significant positive correlations with CRD, R/S, and R/SR. PSSR was negatively correlated with Tchl/Car but positively associated with APX and R/S. RNDVI exhibited a negative correlation with PHR but was positively correlated with Car and Pro. GNDVI demonstrated significant positive correlations with CRD, RV(A) and RDW, while showing negative correlations with Tchl/Car and CAT.PRI displayed a strong positive correlation with Car and Pro but was negatively correlated with PHR and POX. NDRE had significant positive correlations with CRD, DFY, RV(A), and RDW. CRI was negatively correlated with DFY and Tchl/Car but showed positive correlations with APX, POX, and R/S. ARI had a positive correlation with PHR but negative correlations with Car, Tchl, and Pro. GDVI was positively correlated with Car but negatively associated with CAT and POX. Lastly, RGR was positively correlated with RV(A), R/S, and R/SR but negatively correlated with PHR (Table 9).Stepwise multiple linear regression analysis, based on the average data from 2019 to 2020 (Table 10), identified key spectral indices explaining variation in different traits. RARSb and RGR were significant predictors of the root-to-shoot ratio in recovery (r = 0.60%). Similarly, RARSa, PSRI, and ARI accounted for some variation in relative water content (RWC) (r = 0.54%). A combination of WI, PSRI and ARI formed a significant model explaining total chlorophyll variation (r = 0.51%).Table 10 Results from Stepwise regression analysis for predicting some measured traits by spectral reflectance indices in eight tall fescue genotypes during the vegetative stage evaluated under five moisture treatments (C, D1t1D2, D1t2D2, D2 and H2D2) based on the average of two years (2019 and 2020) (n = 80).Full size tablePlant height in recovery was primarily explained by NWI and RGR (r = 0.50%). Additionally, stepwise regression identified RARSb and NDRE as key independent variables contributing to dry forage yield in recovery, explaining a portion of its variation (r = 0.30%) (Table 10).DiscussionThe effect of pollination system on traitsThe observed responses highlight the complex interplay between genetic diversity, drought memory, and SA-induced physiological adjustments, contributing to improved drought resilience. Significant genotypic variation was evident among selfed and open-pollinated populations across multiple morphological, physiological, root traits, and spectral indices. This diversity provides valuable opportunities for selecting genotypes with enhanced drought tolerance. Similar genotypic variation has been reported in other cool-season grasses, such as orchardgrass (Dactylis glomerata) and crested wheatgrass (Agropyron cristatum), where selfing effects varied depending on trait type and environmental conditions24,25,26. These findings align with previous studies, reinforcing the role of genetic diversity in shaping stress tolerance in perennial grasses.In this study, the lack of significant inbreeding depression for biomass, enzymatic antioxidant activities (e.g., APX and POX), and root production potential suggests that selfing may be a viable strategy for developing inbred lines in tall fescue. The improvement of these traits through selfing indicates the fixation of additive genes, reinforcing the potential of recurrent selection to enhance selection gains in tall fescue. Interestingly, previous studies on Dactylis glomerata have reported low heterosis for post-drought recovery-related traits24, which aligns with our findings. It is generally observed that in many annual crops, inbreeding depression reduces fitness-related traits; however, this effect is species-dependent.The effect of severe drought stress on traitsDrought stress is a major limiting factor in plant productivity, affecting physiological and morphological traits. In this study, severe drought stress (D2) significantly decreased plant height (PH), relative water content (RWC), forage yield (WFYand DFY), chlorophyll content (Chla, Chlb, Car, Tchl, and TChl/Car), and antioxidant enzyme activities (CAT and POX). Chlorophyll stability is particularly associated with drought tolerance, as it determines photosynthetic capacity under stress1,27. The reduced forage yield under severe drought stress could be attributed to stomatal closure, which limits CO2 intake and net photosynthesis1,28. These findings align with previous studies on cool-season grasses, where drought-induced reductions in chlorophyll pigments and biomass production were observed1,27,28.A key adaptive mechanism under drought stress is the accumulation of compatible solutes such as proline (Pro). In the present study the increased proline levels under D2 treatment may result from the prevention of proline oxidation or the breakdown of proteins under water deficit conditions29. Man et al.30 demonstrated that increased proline content during drought stress correlates with improved drought tolerance in tall fescue cultivars. Proline plays a critical role in enhancing plant stress tolerance by maintaining osmotic turgor, preventing electrolyte leakage, and stabilizing membranes and enzymes during stress31. However, the relationship between proline accumulation and genetic drought tolerance may vary across different plant species and stress conditions32, highlighting the need for further investigation.The reduction in CAT and APX activities observed under severe is consistent with findings in cool-season turfgrasses1,33. This decline may lead to increased oxidative damage, characterized by elevated lipid peroxidation and impaired physiological functions. The severity of these effects depends on crop species, stress type, duration, and intensity. Our results corroborate previous studies, demonstrating that drought stress reduces antioxidant enzyme activity while increasing proline levels, emphasizing their roles in plant stress response.Root system architecture is a critical determinant of drought resilience, as it influences water uptake and nutrient acquisition. In this study, severe drought stress (D2) significantly enhanced all measured root traits, including root volume (RV(A) and RV(G)), root area (RA), root cumulative length (RCL), root weight (RWW and RDW), root-to-shoot ratio (R/S and R/SR). Under controlled soil moisture conditions, tall fescue plants adapted by spreading their roots more extensively near the surface to optimize water uptake while minimizing energy expenditure34. Concurrently, increased root development at deeper soil layers under drought stress likely facilitated improved water and nutrient acquisition35, suggesting a dual strategy for drought adaptation. These results align with previous studies reporting enhanced root area and volume at deeper soil layers under similar drought conditions23,36,37. The observed increase in the root-to-shoot (R/S) ratio suggests a strategic allocation of resources, where tall fescue genotypes prioritize root growth over shoot development to better withstand water deficits1,22. This adaptive response highlights the critical role of root traits in enhancing drought tolerance.Considering these findings, we propose that root system traits should be integrated into selection indices for breeding programs aimed at improving drought tolerance in tall fescue. By emphasizing root architecture and functionality, breeders can better identify genotypes capable of thriving in water-limited environments, thereby enhancing the overall resilience of tall fescue. These insights contribute to advancing breeding strategies for drought-resistant forage crops, ensuring sustainable agricultural production in changing climatic conditions.Consequence of drought memory on traits Physiological traitsDrought stress memory during early plant growth can be categorized as short-term or long-term. Short-term stress memory typically lasts up to approximately 10 days, whereas long-term stress memory, often regulated by epigenetic mechanisms, may persist throughout the plant’s life and, in some cases, be inherited across generations38. This study investigated drought stress memory by inducing stress at two distinct growth stages (45 days and 90 days after establishment, D1t1D2 and D1t2D2, respectively) providing insights into stage-specific effects on drought memory. Both treatments (D1t1D2 and D1t2D2) significantly enhanced RWC and photosynthetic pigments (Chla, Chlb, Car, and Tchl) compared to the D2 treatment. Improved water retention, as evidenced by enhanced RWC, supports critical physiological processes during subsequent drought periods. Studies in perennial ryegrass have showen that early drought exposure enhances the ability to maintain leaf RWC under recurrent drought conditions39. Similarly, improved photosynthesis during the second stress period may be attributed to drought memory mechanisms, although the underlying processes remain poorly understood40. Drought-induced photoinhibition of photosynthesis is typically associated with stomatal closure, which limits CO2 availability for Rubisco, reducing photosynthetic efficiency41. In contrast, previous studies have reported enhanced photoprotection in drought-preconditioned grasses, though prolonged water deficits can ultimately limit biomass production9.Biochemical traitsOur results revealed stage-specific antioxidant responses: D1t1D2 treatment increased CAT and POX activities, while D1t2D2 treatment elevated Pro and APX activity. This indicates that drought priming at different stages selectively enhances antioxidative defenses, mitigating oxidative damage and improving drought tolerance. Similar findings were reported in previous studies, where drought-primed plants exhibited reduced tissue damage, higher Rubisco levels, increased antioxidative enzyme activities (POX, SOD), and elevated chlorophyll b content42.The interaction between moisture treatments and pollination systems revealed that drought memory was more pronounced in open-pollinated (OP) plants compared to selfed (S1) plants, highlighting the role of genetic diversity in enhancing drought memory. The greater genetic variability inherent in OP plants facilitated stronger physiological and biochemical responses to repeated stress events. In contrast, the S1 system, characterized by reduced genetic diversity, exhibited a weaker drought memory response. Open-pollinated (OP) plants typically display greater genetic diversity due to higher heterozygosity, which may enhance their capacity to adapt to and recover from repeated drought stress events, potentially leading to more pronounced drought stress memory. This increased genetic variability in OP plants likely underpins stronger physiological and biochemical responses to stress. In contrast, selfed genotypes, which exhibit reduced genetic diversity as a result of inbreeding, may show a weaker response to drought stress and consequently less evident drought memory. These differences underscore the importance of genetic diversity in influencing the drought memory response, suggesting that heterozygosity and genetic variability play a critical role in shaping plant resilience under recurrent stress conditions. Further investigation into the mechanisms linking genetic diversity to drought memory could provide valuable insights for improving stress tolerance in Festuca and other species.Furthermore, drought memory was more evident in drought-tolerant genotypes compared to sensitive ones, emphasizing the influence of genetic background on stress adaptation. Tolerant genotypes demonstrated a greater ability to endure and adapt to recurrent stress events. This finding underscores the importance of considering both genetic diversity and genotype-specific traits when evaluating drought resilience. However, our results contrast with previous studies in wheat, where less productive genotypes (in terms of grain rather than biomass) exhibited stronger drought memory responses43,44. This discrepancy may be attributed to differences in species, experimental conditions, or evaluated.Although our study provides valuable insights into drought stress memory, several limitations should be acknowledged. First, the research was conducted under controlled conditions, and field-level variations were not considered, which may influence observed memory effects. Second, the mechanisms linking drought memory to forage yield remain unclear and warrant further investigation. Lastly, the genetic diversity within the experimental genotypes was limited, which could affect the generalizability of the findings. Future research should explore long-term effects, validate findings under field conditions, and incorporate a broader genetic pool to enhance our understanding of drought stress memory and its implications for crop improvement.Effects of salicylic acid on traitsConsistent with previous studies in barley (Habibi45), bluegrass (Azimi et al.46), and ryegrass (Hosseini et al.47), SA treatment has been associated with improved chlorophyll stability under drought stress. This enhancement suggests that SA mitigates photoinhibition and promotes photosynthetic efficiency, which is crucial for sustaining plant growth under water deficit. Additionally, the application of SA led to significant increases in Car, Pro, and antioxidant enzyme activities (CAT and POX) in tall fescue genotypes. This aligns with reports by Ducaiova et al.48 and Pirnajmedin et al.5, who found that exogenous SA application promotes the accumulation of proline, a compound known to mitigate drought-induced damage and protect cellular membranes against oxidative stress caused by reactive oxygen species (ROS). Many studies have further demonstrated that enhancing antioxidant enzyme activities through SA treatment improves the drought tolerance of plants49. Our results strengthen this notion, suggesting that SA plays a key role in enhancing the antioxidant capacity of plants by stimulating carotenoids (Car), catalase (CAT), and peroxidase (POX) under drought stress conditions5. This finding highlights SA as a potential biochemical regulator for improving drought adaptation mechanisms in tall fescue. Moreover, we observed that SA treatment improved RWC, plant height in recovery (PHR), and root-related traits such as such as RDW, R/S, and R/SR. Notably, the interaction between moisture treatments and pollination systems revealed that SA was more effective in S1 genotypes compared to OP ones in terms of the evaluated traits. This suggests that uniform genotypes with fixed additive genes, such as S1, are more likely to benefit from SA treatment. It is possible that this enhanced response is linked to hormonal regulation, gene expression patterns, or metabolic adjustments specific to selfed populations, warranting further investigation.One of the most remarkable findings of this study was the effect of SA on root traits in S1 genotypes. The application of SA under drought stress led to significant improvements in root development, including enhanced root growth, particularly in root tip cell division. Root tip cell division is a crucial process in mitigating the negative effects of water deficit, allowing the plant to better access water and nutrients from the soil. This phenomenon, observed in tall fescue and other species, is a key mechanism by which SA enhances drought tolerance, especially under severe drought conditions47,50. By stimulating cell division in the root tips, SA contributes to more extensive root systems, which are essential for drought resilience. This enhanced root proliferation improves water uptake efficiency and supports overall stress adaptation, reinforcing the role of SA in promoting drought resilience.Overall, these findings suggest that SA not only improves physiological and biochemical responses to drought stress but also plays a significant role in enhancing root development. This, in turn, allows plants to better cope with water limitations and contributes to improved drought tolerance. Integrating SA treatment into breeding and agronomy can enhance drought resilience. Future research should explore its molecular mechanisms and long-term field effects.Selection of genotypesThis study utilized principal component analysis (PCA) to identify superior tall fescue genotypes under varying drought stress conditions. Under the D1t1D2 treatment, PC1 showed positive correlations with photosynthetic pigments, R/S, and R/SR, suggesting higher photosynthetic capacity. Meanwhile, PC2 was positively associated with root traits, indicating an extensive root system. Based on these findings, genotypes 11MOP and 11MS1, characterized by high PC1 and PC2 values, emerged as promising choices for first-time pre-exposure drought stress conditions.For the D1t2D2 treatment, PC1 exhibited negative correlations with dry forage yield in recovery (DFYR), Chla, Tchl, and root characteristics, while PC2 was negatively associated with CAT and APX activities, R/S, and R/SR. Genotypes 11MS1 and 3MS1, both with low PC1 and PC2 values, were identified as tolerant genotypes under this condition.Under D2 treatment, all open-pollinated (OP) genotypes were positioned with high PC2 values, which were correlated with Tchl, Car, POX, CRD, R/SR, and RL. Genotype 11MS1, with low PC1 and high PC2 values, demonstrated suitability for severe drought stress due to its extensive root system and higher levels of Car, POX activity, CRD, and RL.In the H2D2 treatment, PC1 correlated positively with DFY, DFYR, and root characteristics, while PC2 was positively associated with photosynthetic pigments. Genotype 11MS1, characterized by high PC1 and PC2 values, showed a strong positive response to salicylic acid (SA) application, effectively mitigating severe drought stress.Overall, genotype 11 M was identified as a superior choice due to its potential for stress memory under first-time pre-exposure drought stress and its performance under severe drought conditions. Similarly, genotype 11MS1 demonstrated high potential for root and yield production and photosynthetic capacity under simultaneous SA application and drought stress. These findings underscore the utility of PCA as a robust multivariate tool for identifying superior genotypes under complex environmental and treatment conditions, particularly when analyzing multiple interacting traits3,24.PCA effectively differentiates stress-adaptive genotypes, aiding drought resilience breeding. Integrating multiple traits, it refines selection criteria, guiding future genetic studies for improved tall fescue breeding.Spectral reflectance indices (SRIs)In this study, the D2 treatment significantly reduced various spectral reflectance indices (SRIs) such as NDVI, SR, WI, NWI, RARSa, and CRI, compared with the control treatment. These SRIs, derived from visible (VIS) and near-infrared (NIR) wavelengths, are sensitive to physiological traits such as chlorophyll content, leaf color, and water status20,51. For instance, NDVI correlates strongly with leaf chlorophyll content and greenness, while WI and NWI provide insights into the plant’s water status and its ability to maintain moisture during drought stress52,53. Previous studies have shown that under water-limiting conditions, NIR reflectance decreases due to reduced leaf area and chlorophyll content, which in turn impacts photosynthetic capacity and overall plant health54,55.Water-limiting conditions, however, generally result in reduced NIR reflectance and increased visible wavelengths, reflecting faster loss of green leaf area and chlorophyll, which leads to altered SRIs. These alterations are critical as they reflect changes in leaf structure, pigment composition, and overall plant vitality under stress21,23.The foliar application of SA and drought memory in this study helped mitigate the negative effects of water stress, promoting physiological and morphological traits that were detectable using SRIs. Significant effects of genotypes (G), pollination system (P), and their interactions with the five moisture treatments (T) were observed on most SRIs, suggesting that these indices could be valuable tools for selecting genotypes with enhanced drought tolerance. Under the D1t1D2 treatment, for example, WI and RARSb were enhanced in OP, while S1 genotypes showed improved vegetation-SRIs. Similarly, in the H2D2 treatment, WI, RARSa, and CRI were enhanced in OP, while most vegetation-SRIs were improved in S1. These findings suggest that SA and drought memory exert a more pronounced effect in S1 genotypes, improving their drought tolerance54,55.Building on these findings, under the D1t1D2 treatment, improvements were observed in WI, RARSb, ARI, GDVI, and RGR in sensitive genotypes, whereas WI and RGR were enhanced in tolerant genotypes. Similarly, under the D1t2D2 treatment, most vegetation-SRIs improved in both sensitive and tolerant genotypes. Thus, drought memory appeared to be more pronounced in sensitive genotypes compared to tolerant ones based on spectral responses.Our findings indicate that drought memory manifests differently in drought-tolerant and drought-sensitive genotypes, highlighting distinct physiological and spectral mechanisms. In drought-tolerant genotypes, stronger drought memory through physiological traits suggests improved antioxidant defense and water-use efficiency. Conversely, in drought-sensitive genotypes, its manifestation in spectral indices may reflect prolonged stress signals or structural changes in leaf properties, affecting light absorption and reflectance characteristics. Given the absence of conflicting reports, these findings suggest a novel perspective on drought memory in perennial grasses, emphasizing the species-specific and genotype-dependent nature of drought memory. This could have significant implications for breeding programs aimed at improving stress adaptation and drought resilience in tall fescue, highlighting the need to consider both physiological and spectral responses in future selection strategies.Correlation between SRIs with traits and Stepwise multiple linear regression (SMLR)Correlation coefficients are essential statistical tools for assessing the relationships between variables in plant science56, especially when selecting traits linked to drought tolerance. The increasing availability of high-throughput technologies has revolutionized plant breeding, enabling the simultaneous, rapid, and non-destructive assessment of multiple traits. In this study, we explored the potential of spectral reflectance indices (SRIs) as indirect tools for selecting drought-tolerant genotypes in tall fescue breeding programs—a traditionally challenging process often reliant on destructive measurement methods.The phenotypic correlation coefficients between SRIs and various traits revealed several significant relationships. For instance, WI was positively correlated with key photosynthetic pigments (Chla, Chlb, and Tchl/Car) as well as RL and R/SR, suggesting its potential as a marker for genotypes with enhanced photosynthetic efficiency and root production under drought stress. Similarly, NWI showed positive correlations with Tchl/Car and RL, emphasizing its role in identifying genotypes with greater root development and chlorophyll content, both crucial for drought resistance. Furthermore, positive associations were observed between RARSa, PSND, RNDVI and PRI with Car and Pro, supporting the idea that these SRIs are closely linked to biochemical stress responses.Additionally, GNDVI and NDRE displayed strong positive correlations with RV(A) and RDW, while RARSa, RARSb, PSSR, SIPI, PSRI, CRI, and RGR were significantly correlated with R/S and R/SR. These findings underscore the importance of these physiological traits in shaping root architecture, a key factor in drought tolerance.To further investigate the relationships between SRIs and destructive traits, we employed stepwise multiple linear regression (SMLR), treating all SRIs as independent variables. The results showed that RARSb and RGR, both derived from vegetation-specific SRIs, were the most influential indices in explaining variation in root-to-shoot ratio in recovery. Vegetation-SRIs, such as RARSa, PSRI, and ARI, also significantly explained variations in relative water content (RWC), a key physiological indicator of drought tolerance. Furthermore, combined indices from both vegetation and water-SRIs were particularly effective in explaining plant height in recovery and total chlorophyll content. These findings emphasize the utility of combining multiple SRIs from different spectral regions to obtain comprehensive understanding of drought tolerance mechanisms.The stepwise regression analysis also revealed that CRI for dry forage yield and RARSb and NDRE for dry forage yield in recovery were the most significant explanatory variables. This suggests that SRIs, which integrate data from the blue, green, and red-edge regions, are particularly useful for monitoring leaf chlorophyll content, associated pigments, and overall photosynthetic efficiency. In contrast, water-SRIs, which use wavelengths from the near-infrared (NIR) and shortwave-infrared (SWIR) regions, complement these indices by providing insights into plant water status and internal leaf structure, further highlighting their utility in phenotyping drought tolerance traits.These results are consistent with previous studies that demonstrated the effectiveness of SRIs combining information from blue, green, red, and red-edge wavebands for phenotyping various vegetation parameters, such as above-ground biomass and other morphological traits57. Additionally, indices incorporating NIR and SWIR wavebands have proven valuable for estimating yield, water content, and physiological characteristics indirectly, as shown by earlier research58,59.This study provides valuable insights into how SRIs can serve as reliable, non-destructive tools for selecting drought-tolerant genotypes in tall fescue breeding programs. The combination of vegetation and water-SRIs offers a promising approach for enhancing our understanding of the complex physiological and biochemical responses to drought stress. Future research could explore integrating SRIs with genomic tools to better localize the genes or QTLs associated with drought tolerance in this species. Expanding the use of SRIs in field settings could further validate their utility as reliable selection tools for drought resistance.ConclusionsThe findings from this study highlight the significant impact of drought stress on the morphological, physiological, and root functions of tall fescue genotypes, consequently influencing plant growth and biomass production. The observed genetic variation between selfed (S1) and open-pollinated (OP) pollination systems across various traits suggests that altering natural plant mating systems can significantly modify the genetic structure of the germplasm. Pre-exposed to drought at an early stage led to substantial physiological changes, especially in traits like relative water content (RWC), which remained beneficial in later plant stages. Similar trends in measured traits were observed under both pre-exposure drought conditions, with the exception of Chla/b, Tchl/Car, Pro, antioxidant enzymes activities, and RL. Open-pollinated (OP) plants typically display greater genetic diversity due to higher heterozygosity, which may enhance their capacity to adapt to and recover from repeated drought stress events, potentially leading to more pronounced drought stress memory. Further investigation into the mechanisms linking genetic diversity to drought memory could provide valuable insights for improving stress tolerance in Festuca and other species.Additionally, drought memory was more evident in drought-tolerant genotypes, which exhibited faster and more efficient protective responses to recurrent drought conditions. The foliar application of SA enhanced drought tolerance in tall fescue genotypes by promoting physiological traits such as RWC, photosynthetic pigments (Chla, Chlb, and Tchl), carotenoids (Car), enzymatic antioxidant activities (e.g., CAT and POX), proline content, root dry weight, and root-to-shoot ratio.Principal component analysis (PCA) identified genotype 11 M as the superior genotype across all moisture treatments, suggesting its potential utility in breeding programs for the development of synthetic varieties. The comparison of spectral reflectance indices (SRIs) revealed that both water-SRIs (e.g., WI and NWI) and vegetation-SRIs (e.g., RARSa, CRI and RGR) were highly sensitive in explaining key physiological traits. Further research is needed to validate the applicability of these indices across a broader range of genotypes and environmental conditions, to develop a comprehensive model for the rapid screening of drought-tolerant materials in breeding programs.Materials and methodsPlant materials and treatmentsSince 2001, a project on tall fescue has been underway at Isfahan University of Technology. Over the years, 24 tall fescue genotypes were selected from an extensive germplasm collected from various locations in Iran (Isfahan, Yasouj, and Shahroud) and a few other countries (USA, Hungary, and Poland). The genotypes were previously identified through studies that assessed morphological traits, drought tolerance under field conditions, and root and physiological characteristics in pot experiments1,5,22,23. Finally, two drought tolerance (11 M and 21 M) and two susceptible genotypes (1 M and 3 M) were selected (Fig. 3) and planted in the field in February 2017. 789 days after planting, half of the panicles from each genotype (parental plant) were isolated to ensure obligatory selfing, while the remaining half was left uncovered for open pollination. By the end of the summer, seeds from the selfed and open-pollinated panicles were harvested separately, creating two populations: four selfed (S1) progeny and four open-pollinated (OP) progeny (half-sib families). These populations represent contrasting genetic diversity levels, with the OP population maintaining the natural genetic diversity of tall fescue and the S1 population providing a homogeneous group for studying inbreeding depression and trait stabilization.Fig. 3Information of tall fescue genotypes used in the study.Full size imageThe S1 and OP seeds were planted in pots within the research greenhouse at the Isfahan University of Technology in autumn 2019. The experiment was repeated in 2020. The plants were maintained under controlled conditions, with a day/night temperature regime of 25/18 °C, a 14-hour photoperiod, relative humidity of 75 ± 5%, and an average photosynthetically active radiation (PAR) of 400 µmol m–2 s–1. A factorial arrangement based on a completely randomized design with three replications was applied for both years. The experimental factors included: genotypes (8 genotypes), pollination systems (2 levels), and moisture treatments (5 levels).The pot size was selected based on preliminary tests to ensure appropriate root development and space for growth. The pots used for planting were 30 × 16 cm in size, filled with a blend of coarse river sand and silt loam soil in a 1:2 v/v ratio. The weight of soil used in each pot was 7000 ± 50 g, with a pH of 7.6, bulk density of 1.44 g/cm3, and organic matter content of 1.8%. These soil characteristics were chosen to provide a standard growth medium with good drainage and root development potential.Moisture treatments were applied 45 days after seedling establishment (when seedlings reached their full establishment) (Fig. 4). Soil moisture content was monitored gravimetrically by weighing pots before and after irrigation. The total available water depletion was calculated based on the difference in soil weight at field capacity and permanent wilting point. This method ensured that the desired levels of water depletion (40%, 70%, or 90%) were accurately maintained throughout the experiment.Fig. 4Steps of applying five moisture treatments (C, D1t1D2, D1t2D2, D2, H2D2) of tall fescue genotypes during the vegetative stage in this study.Full size image

1.

Control treatment (C): NO limitation on irrigation. Water was supplied when 40% of the total available water in the root zone water was depleted.

2.

Twice drought stress treatment (primary in the first time and secondary at the end stage) (D1t1D2): Primary mild drought stress (70% water depletion) was applied 45 days after establishment for 12 days (D1t1), followed by a recovery period of 90 days with normal irrigation (40% water depletion). Secondary severe drought stress was then applied for 10 days when 90% of the total available water was depleted.

3.

Twice drought stress treatment (primary in the second time and secondary at the end stage) (D1t2D2): Primary mild drought stress (70% water depletion) was applied 90 days after establishment for 12 days (D1t2), followed by arecovery period of 45 days with normal irrigation. Secondary severe drought stress was then applied for 10 days as described in the previous treatment.

4.

Once drought stress treatment (secondary only) (D2): Severe drought stress applied 147 days after establishment for 10 days, with irrigation controlled based on 90% water depletion.

5.

Foliar spray of SA simultaneously with secondary drought stress (H2D2): This treatment was the same as D2, with foliar sparay of salicylic acid (1 mM) applied during the secondary drought stress phase (three times every three dFig. (Fig. 2).

Irrigation of all pots was done depending on the needs of the plant and without considering the type of drought treatment until the complete establishment (about 45 days). Irigation was conducted manually using a graduated cylinder to ensure precise control of the water volume supplied to each pot. The volume of water was calculated based on the weight of soil in each pot and the water-holding capacity of the soil to maintain consistent moisture levels across treatments. During this period, the seedlings were cut three times at two-week intervals to strengthen. Then, primary drought stresses (D1t1 and D1t2) were implemented as mild drought stress at two different stages as explained above (Fig. S1). Experienced plants with primary drought stress, after a rest period (recovery), were treated with secondary drought stress (severe stress) (D1t1D2 and D1t2D2). Once drought stress treatment (D2) was also performed at this stage. Control treatment (C) was subjected to no stress until the end of the experiment. H2D2 treatment was implemented with secondary drought stress and simultaneously salicylic acid (1 mM) was sprayed during stress. After the completion of the mild drought stress phase, some physiological traits (RWC, total chlorophyll, and proline) were assessed to capture the plant’s initial responses, 57 and 102 days after emergence in the D1t1D2 and D1t2D2 treatments, respectively. Morphological (crown diameter, plant height, and forage yield) and physiological (RWC, photosynthetic pigments, proline, and antioxidant enzymes) traits, as well as spectroscopy, were measured immediately following the termination of the severe drought stress phase for all treatments, 157 days after emergence. Morphological (plant height and forage yield) and root characteristics were recorded at the conclusion of the experiment (Fig. S1). It is important to note that all measurements were performed during the vegetative stage and did not coincide with the heading stage or any subsequent reproductive growth phases. This ensured that the observed responses were specific to the vegetative phase and unaffected by transitions into reproductive development.MeasurementsVarious morphological, physiological and Vis-NIR spectroscopy characteristics were measured in this study. Plant height (PH), crown diameter (plant width remaining after cut) (CRD), and wet and dry forage yield per plant (WFY and DFY) were measured following standard methodologies. Leaf water status was assessed by estimating the relative water content (RWC). In addition, spectrophotometry was utilized to quantify total chlorophyll (Tchl), chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car).Furthermore, the proline (Pro) content was assessed using the method outlined by Bates60. It is important to note that enzymatic responses such as Pro, catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POX) are not only part of the physiological responses to drought but also serve as indicators of the impact of drought stress on metabolic processes in plants.For enzyme extraction and assays, 0.1 g of leaves were sampled and frozen in liquid nitrogen. Subsequently, they were ground in a 1 ml solution containing 50 mM phosphate buffer (pH 7.8), 1% polyvinylpolypyrrolidone (w/v), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.2% Triton X-100, 50 mM Tris hydrochloride (Tris–HCL), and 2 mM Dithiothreitol (DTT). The homogenate was centrifuged at 14,000 rounds per minute (rpm) for 30 min, and the resulting supernatant was collected for enzyme assays. Catalase (CAT) activity was determined by monitoring the reduction of absorbance at 240 nm for 2 min following the decomposition of H2O2. Additionally, ascorbate peroxidase (APX) activity was measured by monitoring the decrease in absorbance at 290 nm for 2 min. Peroxidase (POX) activity was assessed by monitoring the increase in absorbance at 470 nm for 2 min. Enzyme activities were expressed per unit protein weight, and protein content was determined using bovine serum albumin as the standard Bradford.For evaluating the root characteristic system, the roots were washed free of soil, and root wet weight (RWW) was measured immediately. Root length (RL), root volume (RV(G)), root area (RA), and root cumulative length (RCL) were measured using a computer scanner and Gia Roots software (version 2009–2011)61. Additionally, root volume (RV(A)) was determined using Archimedes’ law. Root dry weight (RDW) was obtained after drying the roots in an oven at 85 °C for 48 h. The root-to-shoot ratio (R/S) was calculated by dividing the root dry weight (RDW) by the shoot dry weight (SDW), where the SDW corresponds to the dry forage yield (DFY).Diffuse reflectance spectra of tall fescue leaves were acquired using a Vis-NIR photo-diode array spectrometer (model: LR1 spectrometer, ASEQ Instrument, Vancouver, Canada) with a spectral range of 400–1100 nm and a resolution of 0.2 nm, and operating at wavelengths within the visible and near-infrared range. A bifurcated optical fiber (R600-8 Vis-NIR, StellarNet, Inc. Oldsmar, Florida, USA) was employed to transfer light from a 5-W tungsten-halogen lamp powered by rechargeable batteries to the leaf, collect the diffused reflection lights, and pass them to the spectrometer. A black polyamide cylindrical holder was developed in two parts to keep the fiber optic probe in a fixed position relative to the leaf samples, create a controlled environment to minimize the impact of ambient light and facilitate the collection of dark and reference spectra. The exposure time and the number of scans were set as 800ms and 5, respectively. Dark and reference spectra were acquired before every 30 spectral measurements to remove the effects of external lighting and obtain the relative spectra of the leaf samples. Three leaves of each genotype and two parts of each leaf, finally six diffuse reflectance spectra for each genotype, were measured and the averaged spectrum of six spectra was used for subsequent analysis. All measurements were conducted in the greenhouse without removing the tall fescue leaves. The relative reflectance spectrum was then used in the calculation of eighteen different indices used in this study, which are listed in Table S1.Statistical analysisAfter assessing the normality of the data using the Q-Q plot test, the data were subjected to analysis of variance (ANOVA) using SAS 9.462 to assess differences between the years, genotypes, pollination systems, moisture treatments, and their interactions for each variable. Mean comparisons for different traits and spectral reflectance indices were conducted using the LSD test (P < 0.05). Correlation coefficients between spectral reflectance indices and various traits were computed using the proc CORR function in SAS. Principal component analysis (PCA) was conducted using Stat Graphics Centurion XVII to reduce the dimensionality of the data space based on a correlation matrix56,63. A stepwise multiple linear regression analysis was performed using SAS 9.462 to identify the spectral reflectance indices that significantly contributed to the variation in different traits.

Data availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

AbbreviationsCRD:

Crown diameter

PH:

Plant height

WFY:

Wet forage yield

DFY:

Dry forage yield

PHR:

Plant height in recovery

WFYR:

Wet forage yield in recovery

DFYR:

Dry forage yield in recovery

RWC:

Relative water content

Chla:

Chlorophyll a content

Chlb:

Chlorophyll b content

Car:

Carotenoid content

Tchl:

Total chlorophyll

Chl a/b:

Ratio of Chla/Chlb

Tchl/Car:

Ratio of Tchl/Car

Pro:

Proline content

CAT:

Catalase activity

APX:

Ascorbate peroxidase activity

POX:

Reroxidase activity

RL:

Root length

RV(A):

Root volume (Archimedes)

RV(G):

Root volume (Giaroot)

RA:

Root area

RCL:

Root cumulative length

RWW:

Root wet weight

RDW:

Root dry weight

R/S:

Root-to-shoot ratio

R/SR:

Root-to-shoot ratio in recovery

NDVI:

Normalized difference vegetation index

SR:

Simple ratio

WI:

Water index

NWI:

Normalized water index

RARSa:

Ratio analysis of reflectance spectra

RARSb:

Ratio analysis of reflectance spectra

PSSR:

Pigment specific simple ratio

PSND:

Pigment specific normalized different

SIPI:

Structure intensive pigment index

RNDVI:

Red normalized difference vegetation index

GNDVI:

Green normalized difference vegetation index

PRI:

Photochemical reflectance index

NDRE:

Normalized difference red edge index

PSRI:

Plant senescence reflectance index

CRI:

Cartenoid reflectance index

ARI:

Anthocyanin reflectance index

GDVI:

Green difference vegetation index

RGR:

Red/ green ratio

SRIs:

Spectral reflectance indices

OP:

Open-pollinated

S1

:

Selfed

C:

Control treatment

D1t1

:

Primary mild drought stress in first time

D1t2

:

Primary mild drought stress in second time

D1t1D2

:

Twice drought stress treatment (primary in the first time and secondary at the end stage)

D1t2D2

:

Twice drought stress treatment (primary in the second time and secondary at the end stage)

D2

:

Once severe drought stress treatment (secondary only)

H2D2

:

Foliar spray of SA simultaneously with secondary drought stress

G:

Genotype

P:

Pollination system

T:

Treatment

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Statgraphics Statgraphics Version 15.2.11 (Statpoint Technologies: The Plains, 2007).Download referencesAcknowledgementsThe authors thank Isfahan University of Technology (IUT) and Mobarakeh Steel Technology and Innovation Development company (MSTID) to support this work.Author informationAuthors and AffiliationsDepartment of Agronomy and Plant Breeding, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, IranMaryam Safari & Mohammad Mahdi MajidiAuthorsMaryam SafariView author publicationsYou can also search for this author in

PubMed Google ScholarMohammad Mahdi MajidiView author publicationsYou can also search for this author in

PubMed Google ScholarContributionsMS Investigation; Methodology; Project administration; Software; Writing original draft. M M M: Conceptualization; Funding acquisition; Resources; Supervision; Validation; Review & editing.Corresponding authorCorrespondence to

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Reprints and permissionsAbout this articleCite this articleSafari, M., Majidi, M.M. Role of genetic diversity and salicylic acid in drought stress memory of tall fescue.

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KeywordsTall fescueDrought stressStress memorySalicylic acidSpectral reflectance indices (SRIs)Genetic variation.

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