MADS-box gene expression patterns correlate with drought and salt stress responses in Kentucky bluegrass (Poa pratensis L.)
Komal Tariq, Xue You, Yifeng Jin, Haoran Yu, Sabir Iqbal, Adil Hussain, Yang Chen
TL;DR
This study explores how MADS-box genes in Kentucky bluegrass respond to drought and salt stress, identifying potential roles in stress tolerance.
Contribution
The study identifies 16 MADS-box genes in Kentucky bluegrass and reveals their tissue-specific expression patterns under drought and salt stress.
Findings
Arcadia variety showed greater tolerance to drought and salt stress compared to other varieties.
12 out of 16 MADS-box genes exhibited tissue-specific expression under drought stress.
Six MADS-box genes were significantly upregulated in all tissues under salt stress.
Abstract
Kentucky bluegrass (Poa pratensis L.) is a perennial cool-season turfgrass commonly utilized in lawns. However, turfgrass quality is affected by abiotic stress. Guided by previous reports demonstrating the critical involvement of MADS-box transcription factors in abiotic stress responses in monocot species such as rice (Oryza sativa) and sheepgrass (Leymis Chinesis), this study specifically focuses on the MADS-box gene family to investigate their potential roles in stress adaptaion mechanisms in Kentucky bluegrass. Although MADS-box genes are well-characterized as regulators of plant growth and development, there is currently limited information on their stress-related functions in this economically important turfgrass species. This study examined the physiological and molecular responses of three Kentucky bluegrass varieties (K.B.G., Arcadia, and Jenny) to salt and drought stress.…
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Figure 10- —National Natural Science Foundation of China
- —Natural Science Foundation of Heilongjiang Province
- —Fundamental Research Funds in Heilongjiang Provincial Department of Education
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Taxonomy
TopicsTurfgrass Adaptation and Management · Plant Stress Responses and Tolerance · Plant Molecular Biology Research
Introduction
The MADS-box family of transcription factors (TFs) encodes master regulators of gene expression across the different kingdoms of life (Fujinaga, Huang & Peterlin, 2023). They are mostly linked to the development of plants, metabolism, and stress management (Ma et al., 2023). MADS TFs constitute a large and extensively studied TF family in eukaryotes, including plants, animals, and fungi (Liang et al., 2023; Ma et al., 2021). They play important roles in controlling multiple growth processes of plants, including flower, embryo, and fruit development, and the growth of vegetative organs (Shah et al., 2022). MADS is an acronym for mini chromosome maintenance 1 (yeast), Agamous (Arabidopsis thaliana), Deficient (snapdragon), and serum response (Humans). MADS-box proteins comprise four different functional domains, ranging from the N to the C terminus: the MADS-box (M), intervening (I), keratin like (K), and C-terminal (C) domains. A highly conserved DNA-binding domain, called the MADS-box domain (M), in the protein’s N-terminal region is roughly 58–60 amino acid residues long, a characteristic shared by all MADS-box genes. The M domain allows nuclear localization, dimerization with other MADS-box proteins, and binding to CC(A/T)rG boxes in the regulatory regions of their target genes (Aerts et al., 2018). It is distinguished by a coiled-coil structure made up of three amphipathic helices and mostly aids in heterodimerization (Zhang et al., 2023). The least conserved domain, the intervening (I) domain, which is approximately 30 amino acids long and triggers the dimerization of protein and DNA-binding specificity, lies between the K domain and the MADS-box (Shen, Jia & Wang, 2021). The development of higher-order multimeric MADS-box protein complexes and transcriptional activation depend on the C-terminal (C) domain, which varies in length and sequence. It is primarily composed of hydrophobic amino acids (Chen et al., 2019).
Phylogenetic analysis further categorizes the MADS-box proteins into Type-1 and Type-II (MIKC-type genes) (Qu et al., 2021). Type-1 MADS-box proteins are further distinguished into three subgroups: Mα, Mβ, and Mγ (Callens et al., 2018; Zhang et al., 2021). These TFs produce shorter proteins, which are characterized primarily by the MADS-box domain, a conserved region. Currently, very few Type-1 genes have been studied, but play a key role in plant reproductive processes, including embryogenesis and gametophyte development, helping to regulate various stages of reproduction (Hoffmann et al., 2022; Zhang et al., 2025). Research has shown that the expression of certain Type I components varies notably under stress conditions, which may contribute to the plant’s stress response mechanisms. However, a more extensive and detailed study has been conducted on Type II MADS-box genes (Shah et al., 2022) and are mainly involved in plant development and stress responses (Agarwal & Khurana, 2019). Recent studies have suggested that the Type II MADS-box genes are crucial for plants to withstand extreme environmental conditions such as salt, drought, heat, and cold (Dong et al., 2021). Type II MADS-box genes, also referred as MIKC type MADS genes, are distinguished by their numerous exons and introns (Zhao et al., 2022). These are further divided into two subgroups (MIKC* and MIKC^c^) (Kumpeangkeaw et al., 2019). The MIKC^C^ genes are divided into about 13 subfamilies, the majority of which are derived from ancient seed plants, and their C-terminal regions frequently exhibit unique sequence motifs, which are linked to functional diversification across subfamilies: AGAMOUS, APETALA1, AGAMOUS-LIKE 12, AGAMOUS-LIKE 15, AGAMOUS-LIKE 17, B-SISTER, DEFICIENS (DEF)/GLOBOSA, FLOWERING LOCUS C, SEPALLATA, SQUAMOSA, SHORT VEGETATIVE PHASE, and TM3/SOC1 (Díaz-Riquelme et al., 2009).
However, further studies revealed that abiotic stresses, including salt, cold, and drought stress, may impact the expression of some MADS-box genes (Masiero et al., 2011). For example, in rice, seven MADS-box genes showed variable expression in response to salt and drought stresses, and three of these genes were shown to be down-regulated (Zhang et al., 2025) when subjected to salt and drought stress (Arora et al., 2007). Also, upregulation of the OsMADS26 gene in rice has been associated with severe stress sensitivity. Further investigations have revealed that OsMADS26 may negatively influence drought tolerance and disease resistance (Khong et al., 2015). Similarly, abiotic stresses (drought and salt) increased the expression of the AGL2 gene in wheat (Zhang et al., 2012). Additionally, it was reported that abiotic stresses such as salt, wounding, and dehydration rapidly induced the expression of SlMBP11, an AGL15-like MADS-box gene in Solanum lycopersicum (Guo et al., 2016). Moreover, several plant hormones also activated SIMBP11, which may participate in abscisic acid (ABA)-independent signaling networks regulating salt-stress responses (Castelán-Muñoz et al., 2019). In addition to hormonal signaling and gene regulation in response to salt stress, plants also develop physical defenses like cuticular wax to help reduce the impact of environmental challenges.
The outermost hydrophobic layer of aerial plant tissues, called cuticular wax, serves as a barrier from biotic and abiotic environmental stresses and inhibits excessive non-stomatal evaporation (Yeats & Rose, 2013). Ecological factors and developmental processes have an impact on wax accumulation (Shepherd & Wynne Griffiths, 2006). Cuticular wax has a varied composition and structure; it has been known to indicate resilience to various adverse environmental conditions and has been suggested as a potentially advantageous trait (Dhanyalakshmi et al., 2019). During drought stress, the amount of cuticular wax increases in a variety of plants, including Arabidopsis, Camelina sativa, potato, barley, maize, tomato, eggplant, and cabbage (Shaheenuzzamn et al., 2019). Additionally, the number of plant species, including Zea mays, Triticum aestivum, Glycine max, Pinus palustris, Avena sativa, Nicotiana glauca, Gossypium hirsutum, and Sesamum indicum, increases the amount and composition of wax up to 2.5-fold in their leaves after 4 h during drought stress (Cameron, Teece & Smart, 2006; Kosma & Jenks, 2007; Shepherd & Wynne Griffiths, 2006). Another study described that cuticular wax composition increased up to 2.6-fold under drought conditions (20% PEG 6,000 treatment) in wheat (Wang et al., 2015), and up to 90% in sorghum (Shaheenuzzamn et al., 2021). Cuticular wax’s thickness and composition can vary from plant to plant and under different environmental conditions (Tipple & Pagani, 2013). To face different stresses, plants have developed a network of integrated molecular and cellular responses, including leaf orientation (Xue et al., 2017), cuticular wax thickening (Singh, Das & Geeta, 2018), and efficient stomatal regulation and structure (Chen et al., 2017; Pornsiriwong et al., 2017).
Kentucky Bluegrass (Poa Pratensis L.) is a rhizomatous member of a significantly important group of grass species with considerable economic and agronomic importance (Braun et al., 2022). It is a considerably expensive cool-season turfgrass that is utilized in home lawns, golf courses, and public park regions with moderate and cold-temperate climates. This grass has numerous improved cultivars that are utilized in landscaping all over the world (Honig et al., 2012). Despite having significant ornamental value, Kentucky bluegrass is severely limited in its use due to water shortages, particularly in arid and semiarid regions, as well as those areas where the need for water is rising for residential, industrial, and agricultural purposes (Fan et al., 2020). Choosing water-saving cultivars and lowering water input in grass maintenance has been challenging (Serba et al., 2022). Abiotic stress affects the texture, shoot density, uniformity, color, and growth habit of Kentucky bluegrass, which reduces the quality of turf (Saud et al., 2014, 2016). Diverse varieties of Kentucky bluegrass react to abiotic stress by using distinct adaptive approaches and mechanisms based on their physiology, molecular basis, and morphology. Meanwhile, the performance of Kentucky bluegrass can be challenged by abiotic stresses such as salt and drought stress, and there has been limited focus on abiotic stresses in bluegrass.
In this study, we studied the physiology of Kentucky bluegrass and identified the MADS-box transcription factor genes from the transcriptome analysis of P. pratensis to investigate their expression under salt and drought stress. Our approach aims to provide insight into the potential involvement of MADS-box genes in stress responses, while recognizing that further functional studies are necessary to confirm their causal role in stress adaptation.
Materials and Methods
Plant material and growth conditions
The experiments were conducted on one-year-old Kentucky bluegrass varieties (K.B.G., Arcadia, and Jenny). These varieties were obtained from Clover Ecological Technology Co., Ltd., Beijing, China. Arcadia is a compact, fine-textured cultivar selected for superior performance and field use, with high turf quality and reported drought tolerance characteristics (Saud et al., 2020). Although specific published data for K.B.G. and Jenny cultivars under stress are limited, Kentucky bluegrass cultivars have been shown to vary significantly in their drought and salt stress responses, with some cultivars demonstrating relatively better tolerance and recovery under imposed stress conditions. Therefore, these varieties were selected to represent physiological variation within Kentucky bluegrass, allowing comparative analysis of physiological responses to abiotic stress.
The varieties were planted in Qiqihar University’s nursery greenhouse located in Qiqihar City, Heilongjiang Province, China, in nutrient soil (loam: vermiculite: sand ratio is 3:1:1). The growth conditions in the greenhouse were maintained at a temperature of 24 °C during the day and 15 °C at night, with a 13-h light/11-h dark photoperiod. The light intensity was set at 500 µmol/(m^2^·s), and the relative humidity was kept at 60%. A summary of the experimental workflow and the major results obtained from each step is presented in Fig. S1.
Drought and salt stress
To better understand the impact of drought stress induced by withholding water on the physiology of Kentucky bluegrass, nursery plants of three bluegrass varieties (K.B.G., Arcadia, and Jenny) were pruned and transplanted into nine uniform pots (18 cm deep and 10 cm diameter), with three replicates per variety, and five plants per pot. Each pot was filled with a consistent soil substrate. Prior to stress imposition, plants were watered to full soil saturation and maintained under well-watered conditions for a 2-week acclimation period to ensure uniform establishment. Drought stress was subsequently imposed by withholding irrigation for 21 days, following a commonly used soil-drying approach (Luo et al., 2011). Polyethylene glycol (PEG) was not applied in this soil-based experiment, as the aim was to simulate natural drought stress under pot conditions. Although soil moisture content and water potential were not quantitatively measured, drought severity was monitored through visible phenotypic symptoms, including leaf rolling, wilting, and growth inhibition. Plant images were recorded at 0, 7, 14, and 21 days to document stress progression. For molecular analysis, a separate drought stress treatment was applied to one-year-old plants of one variety (Arcadia). To minimize root damage during uprooting, we first watered the soil to soften it, making it easier to extract the plants carefully. After uprooting, the roots were cleaned with the help of water and dipped in nutrient solution (1/2 MS) to hydrate the roots, avoid nutrient deficiency, and standardize conditions, ensuring the plants were in optimal condition for the experiment. The plants were then treated with a 10% polyethylene glycol-6000 PEG treatment to induce drought stress (Shereen et al., 2019). Leaves and stem sections were collected after 0, 2, 6, and 16 h for real-time polymerase chain reaction (PCR) analysis.
Salt stress under soil conditions was applied to the same three varieties (K.B.G., Arcadia, and Jenny) using an experimental design identical to that of the drought assay. Plants were irrigated with 400 mM NaCl solution every two days for 20 days after acclimation. This concentration was selected to induce clear salinity stress symptoms within a short experimental period, allowing comparative evaluation of varietal tolerance. Stress progression was assessed based on visible symptoms such as leaf chlorosis, growth retardation, and wilting. Images were captured at 0, 10, and 20 days. Similarly, for molecular analysis, one-year-old uniform soil-grown plants of one variety (Arcadia) were uprooted and carefully washed as described above. The plants were then immersed in 1/2 MS medium containing 0, 100, 150, or 200 mM NaCl, with these values referring to the NaCl concentrations in the solution in which the plants were immersed, and 0 mM serving as the no-salt control. Subsequently, leaf and root sections were collected after 7 days for real-time PCR. Real-time PCR samples were kept at −80 °C after a liquid nitrogen snap-frozen process.
Chlorophyll content
The chlorophyll content was measured using the ethanol extraction method (Aono et al., 2021; Hiscox & Israelstam, 1979). After weighing 0.2 g of Kentucky bluegrass stems and leaves, 10 ml of 95% ethanol was added to the samples. The mixture was then left to extract at room temperature for 48 h in the dark. After a quick centrifugation of the extracts, the supernatants were obtained for further analysis. The absorption of the extracted mixture was quantified at 663 and 645 nm in a UV spectrophotometer (MACY-UV1100; Macylab, Shanghai, China) in triplicate. Chlorophyll content was measured with the formula:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $Total{\rm \; }Chlorophyll{{\rm \; }_{\left( {mg/g{\rm \; }fresh{\rm \; }weight} \right)}}{\rm \; } = \left( {20.08 \times {A_{645}} + 8.02 \times {A_{663}}} \right) \times \displaystyle{{Vol} \over {W \times 1\!,\!000}}$\end{document}where;
V stands for the volume (in ml) of the sample
W is the weight (g) of the tissues.
Scanning electron microscopy analysis of stem and leaf epidermis
The epidermal structures of leaves and stems of the three varieties of Kentucky bluegrass were examined using scanning electron microscopy (Cui et al., 2023). This analysis was conducted under non-stress conditions to ensure a comprehensive evaluation of the structural differences among three varieties (K.B.G., Arcadia, and Jenny). Fresh leaves and stem sections (5 mm^2^) were collected and fixed immediately for 24 h at room temperature. Next, the fixed specimens were washed with 0.1 M phosphate buffer (pH 7.4) three times (15 min every single time) and fixed again with 1% osmic acid in 0.1 M phosphate buffer (pH 7.4). After brief washing, the fixed specimens underwent 15 min of moderate alcohol dehydration using a gradient of 30%, 50%, 70%, 80%, 90%, 95%, 100%, and 100%. After 15 min of isoamyl acetate soaking, they underwent critical point drying. Dual-sided conductive carbon film adhesive tape was used for holding the dry samples and sprayed with gold for 30 s (IXRFmodel550i). The specimens were inspected with a scanning electron microscope (SU8010; Hitachi, Tokyo, Japan).
Transcriptome-based identification and differential expression screening of MADS-box genes
To systematically identify MADS-box transcription factor genes in Kentucky bluegrass, transcriptome datasets of three varieties (K.B.G., Arcadia, and Jenny) were generated (NCBI BioProject: PRJNA1217998). Raw paired-end reads were quality-filtered using fastp with default parameters (Chen et al., 2018), and clean reads were subjected to de novo transcriptome assembly using Trinity (Grabherr et al., 2011).
For MADS-box gene identification, the assembled transcripts were first annotated using Diamond BLASTX against the NCBI non-redundant (Nr) protein database and Swiss-Prot database with an E-value threshold of ≤1.0 × 10^−5^. Similarly, hidden Markov model (HMM) searches were performed using HMMER v3.3 (http://hmmer.org/) against the Pfam database (Pfam ID: PF00319, MADS-box domain) to identify transcripts encoding putative MADS-box proteins. Candidate sequences were further validated by confirming the presence of the conserved MADS-box domain (approximately 58–60 amino acids) using the NCBI Conserved Domain Database (CDD) and SMART (http://smart.embl-heidelberg.de/).
Through this combined annotation pipeline, a total of 33 putative MADS-box transcripts were identified in the Kentucky bluegrass transcriptome. Gene expression levels were quantified as Transcripts Per Million (TPM), and differentially expressed genes (DEGs) among the three varieties were identified using DESeq2 (Love, Huber & Anders, 2014), with significance thresholds of |log₂(fold change)| > 1 and adjusted q-value < 0.05. Among the 33 identified MADS-box transcripts, 26 (78%) transcripts exhibited significant differential expression across different varieties and were identified as 16 distinct genes. Therefore, these genes were selected as subjects for subsequent functional characterization studies.
The selection of MADS-box genes as the focal gene family in this study was based on two complementary considerations: (1) biological evidence from prior studies demonstrating the critical involvement of MADS-box transcription factors in abiotic stress responses in grasses such as rice (Oryza sativa) and sheepgrass (Leymus chinensis) (Arora et al., 2007; Jia et al., 2018; Wei et al., 2018); and (2) statistical identification of differentially expressed MADS-box members from our transcriptome data. This hypothesis-driven yet data-validated approach ensures that the candidate genes investigated herein possess both biological plausibility and empirical support for stress-related functions.
Data collection parameters
The phenotypic effects of drought stress (withholding water) on plants were recorded after 7 days and up to 21 days, and high-concentration salt stress effects were recorded from 10 to 20 days. The molecular effects of drought treatment were identified at 0, 2, 6, and 16 h, whereas salt treatment effects were recorded with concentrations of 0, 100, 150, and 200 mM after 7 days.
RNA extraction and cDNA synthesis
Total RNA was obtained from sections of leaves, stems, and roots by RNAprep Pure Plant Kit (TIANGEN, Beijing, China), following the manufacturer’s instructions for later use in qRT-PCR analysis. The RNAs were examined via agarose gel electrophoresis and via DS-11FX+ (DeNovix, Wilmington, DE, USA) to assess their purity, integrity, and concentration. Following that, 1 µg of RNA was used for cDNA synthesis using the HiFiScript cDNA synthesis kit (CWBIO, Taizhou, Jiangsu, China) following the manufacturer’s instructions.
Quantitative real-time PCR analysis
The expression levels of 16 genes in leaf and stem tissues subjected to different salt and drought treatments were investigated using quantitative real-time PCR, using the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). A total of 12.5 µl of TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (No. RR820A; Takara, Dalian, China), 2 µl (100 ng) of cDNA, 1 µl of each forward and reverse primer, and 8.5 µl of RNase-free water were used to set up 25 µl reactions for this purpose. After 30 s at 95 °C, the program ran 40 cycles, each consisting of 5 s at 95 °C and 20 s at 68 °C. UBQ was used for the internal control in three technical as well as three biological replicates. Primers were designed using Primer Premier 5.0 software. The expression level of each gene has been examined using the 2^−ΔΔCT^ technique (Schmittgen & Livak, 2008). For normalization and comparison, expression levels at 0 h and 0 mM (pre-treatment) were used as the baseline control for each gene. Additional file 1: Table 1 contains the list of primers used in the current study.
Prediction of cis-acting elements in promoter sequences
To predict the cis-acting elements, 2,000 bp regions upstream of the translational start codon have been examined as promoters. All of the promoter sequences were downloaded from NCBI and were uploaded to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for the verification of cis-acting elements. The figure was created utilizing the function and location of many cis-acting elements using TBtools software (Chen et al., 2020).
Statistical analysis
All statistical analyses were conducted using IBM SPSS Statistics (version 25.0; IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation (SD) from at least three independent biological replicates unless otherwise specified. Prior to analysis, data were tested for normal distribution and homogeneity of variance; where assumptions were not met, appropriate data transformation was applied.
For physiological parameters (chlorophyll content) measured across three varieties (K.B.G., Arcadia, and Jenny) at multiple time points under drought (0, 7, and 14 days) or salt stress (0, 10, and 20 days), the significance of differences was assessed using Tukey’s Honestly Significant Difference (HSD) test, with family-wise error rate controlled at α = 0.05. Pairwise comparisons were performed among varieties within each time point and among time points within each variety. For comparisons involving two groups (e.g., control vs. stress within a single time point or treatment), Student’s t-tests were used.
For qRT-PCR experiments, the relative expression levels of 16 MADS-box genes were quantified using the 2^−ΔΔCt^ method (Schmittgen & Livak, 2008), with UBQ as the internal reference gene. Expression data were normalized to the untreated control (0 h for drought stress; 0 mM for salt stress) for each gene. (i) Drought stress time-course experiments: Gene expression was measured in leaves and stems at four time points (0, 2, 6, and 16 h). For each tissue type, differences in expression levels across time points were evaluated using Tukey’s HSD test. (ii) Salt stress concentration-gradient experiments: Gene expression was measured in leaves and stems after 7 days of exposure to four NaCl concentrations (0, 100, 150, and 200 mM). Similarly, Tukey’s HSD test was used to compare expression levels across concentration gradients within each tissue type. For both drought and salt stress experiments, analyses were performed separately for each tissue (leaves vs. stems) to focus on tissue-specific expression patterns. Given the exploratory nature of this study involving 16 candidate genes, we applied the Benjamini-Hochberg false discovery rate (FDR) correction to control for multiple testing, with adjusted q-values < 0.05 considered statistically significant.
For transcriptome-based identification of differentially expressed genes (DEGs), expression levels were quantified as TPM. DEGs among the three varieties were identified using DESeq2 (Love, Huber & Anders, 2014), with significance thresholds of |log2(fold change)| > 1 and Benjamini-Hochberg adjusted q-value < 0.05 (Love, Huber & Anders, 2014).
Statistically significant differences are indicated by different lowercase letters in the figures, where groups sharing the same letter are not significantly different; asterisks are used to denote significance levels (*p < 0.05; **p < 0.01). Error bars represent ± standard deviation from three biological replicates, each with three technical replicates.
Results
Comparing the response of bluegrass varieties to drought and salt stress
Three varieties (K.B.G., Arcadia, and Jenny) of Kentucky bluegrass were subjected to drought stress (withholding water) and salt treatment (Fig. 1). At 7 days of drought stress, Jenny’s leaves began to show the effect of drought stress, characterized by chlorosis and wilting. After 14 days of stress, these drying effects further intensified, especially on the leaves of Jenny plants, followed by obvious chlorosis and wilting of K.B.G. leaves. On the other hand, the Arcadia plants grew relatively well for the same period. When the stress time reached 21 days, Jenny plants were completely dried, whereas Arcadia and K.B.G. still had some green tissues left (Fig. 1A). Similar differences in the amount of chlorophyll were reported. Measuring the chlorophyll content under drought stress at 0, 7, and 14 d found that the chlorophyll content at 0 d of Jenny plants was lower than that of K.B.G. and Arcadia plants. Arcadia’s chlorophyll concentration was noticeably higher than K.B.G. and Jenny after 7 days. The chlorophyll content of Jenny was significantly lower than that of the other two varieties after 14 days of drought conditions, while there was no significant difference between Arcadia and K.B.G. (Fig. 1B).
Analysis of drought resistance in three varieties.(A) Wilting effects of ‘K.B.G., Arcadia and Jenny’ with different intervals of time; (B) Difference in chlorophyll content of ‘K.B.G., Arcadia and Jenny’ at 0, 7 and 14 d under stress. Significant differences are shown by asterisks () according to the Student’s T-test at a 0.05% level of significance. The standard error is represented by error bars and ns = non-significant.*
All the varieties appeared to have the same impact of NaCl for up to 10 days. However, the effects of salt stress were more pronounced on Jenny plants, characterized by yellowing and narrow leaves, followed by K.B.G. after 20 days of salt treatment, while Arcadia plants were relatively well (Fig. 2A). Meanwhile, the chlorophyll content of the three varieties under salt treatment remained the same as that of drought stress (Fig. 2B). Overall, the chlorophyll content of Arcadia was consistently the highest compared to the other two varieties, followed by K.B.G. and Jenny. Arcadia exhibited greater resistance compared to other varieties and was therefore selected for subsequent experimental analyses.
Analysis of salt resistance in three varieties.(A) Wilting effects of ‘K.B.G., Arcadia and Jenny’ with different intervals of time; (B) difference in chlorophyll content of ‘K.B.G., Arcadia and Jenny’ at 0, 10 and 20 d under treatment, significant differences are shown by asterisks () according to Student’s T test at a 0.05% level of significance. The standard error is represented by error bars and ns = non-significant.*
Microstructures of leaf lower and upper epidermis
To explore potential anatomical differences among the three Kentucky bluegrass varieties, we examined the micromorphology of leaf epidermal surface using scanning electron microscopy (SEM). Scanning electron microscope images of the leaves of three varieties of Kentucky bluegrass showed that there were significant variations in the ultrastructure of both the upper and lower epidermis of the three varieties. The wax attachment density in the upper epidermis was significantly greater than that of the lower epidermis (Figs. 3A–3F), and the lower epidermis was smoother than the upper epidermis (Figs. 3G–3I). Wax density of the lower epidermis of K.B.G. and Jenny was not observed in the images, whereas some wax crystals have appeared on the lower epidermis of Arcadia (Figs. 3G–3I). The stomatal area of the Arcadia was also significantly higher than Jenny and K.B.G. (Figs. 3J–3L). These structural variations, especially the higher density of wax on Arcadia’s lower epidermis, may improve its ability to retain moisture and control water loss in stress conditions, potentially contributing to its observed tolerance to both drought and salt stress compared to the other varieties.
SEM images of leaf upper and lower epidermis of ‘K.B.G., Arcadia and Jenny’.(A–C) The wax density of ‘K.B.G., Arcadia and Jenny’ upper epidermis under 10,000x; (D–F) The wax density of ‘K.B.G., Arcadia and Jenny’ upper epidermis under 20,000x; (G–I) The wax density of ‘K.B.G., Arcadia and Jenny’ lower epidermis under 500x; (J–L) The wax density and stomata of ‘K.B.G., Arcadia and Jenny’ lower epidermis under 3,000x.
Microstructure of stem epidermis
SEM analysis of stems of three Kentucky bluegrass varieties revealed considerable significant differences in stem structure among the three varieties. Arcadia had the highest epidermal papillae density, followed by K.B.G., and Jenny (Figs. 4A–4C) showed the lowest density. It is worth noting that the papillae morphology of the three varieties was also different (Figs. 4D–4F). For stomata, the area of the three varieties was K.B.G.>Arcadia>Jenny (Figs. 4G–4I). These distinctive anatomical features, particularly the higher papillae density, may contribute to differences in stress defense mechanisms. These observations were intended as preliminary morphological screening to visually identify traits that might correlate with stress tolerance, rather than as definitive quantitative measurements.
SEM images of stem epidermis of ‘K.B.G., Arcadia and Jenny’.(A) (a–c) The number of stomata and papillae on stem epidermis of ‘K.B.G., Arcadia and Jenny’ under 200x; (d–f) Structural differences of ‘K.B.G., Arcadia and Jenny’ papillae under 1,500x, (g–i) Differences in total stomatal area of ‘K.B.G., Arcadia and Jenny’ under 3,000x. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation. (B) Graphs showing the number of papillae, and stomata.
Transcriptome-based identification and expression profiling of MADS-box genes
Transcriptome analysis of three Kentucky bluegrass varieties identified multiple transcription factor families, including NAC, MYB, WRKY, and MADS-box (Fig. 5A). Given the well-documented roles of MADS-box transcription factors in mediating abiotic stress tolerance in monocot species such as rice and sheepgrass (Arora et al., 2007; Castelán-Muñoz et al., 2019; Jia et al., 2018), we hypothesized that MADS-box genes might similarly contribute to drought and salt stress responses in Kentucky bluegrass. To test this hypothesis, we employed a systematic screening strategy. Using HMM-based domain searches (Pfam: PF00319) combined with BLAST annotation, a total of 33 MADS-box transcripts were identified in the assembled transcriptome. Differential expression analysis revealed that 26 of these 33 transcripts displayed statistically significant variation in expression among varieties (|log_2_FC| > 1, q < 0.05), suggesting potential functional divergence related to stress adaptation (Fig. S2), these transcripts were identified as 16 genes. Gene Ontology (GO) enrichment analysis indicated that these 16 genes were predominantly associated with transcriptional regulation, including positive regulation of transcription by RNA polymerase II and cis-regulatory region binding (Fig. 5B).
Summary of the transcriptomic results.(A) List of transcription factors from transcriptome analysis. (B) GO enrichment analysis of transcription factors. The *** (triple asterisks) indicate highly significant enrichment (p-adjust < 0.001). (C) Heatmap showing the MADS-gene box family’s expression profile among three varieties. Red and blue indicate different levels of gene expression, from high to low, and white displays median expression.
Expression heatmap analysis demonstrated distinct transcriptional profiles of the 16 MADS-box genes across K.B.G., Arcadia, and Jenny (Fig. 5C). In K.B.G., genes including AGL28, AGL61, AGL62, MADS16, MADS26, MADS51, and MADS57 showed elevated expression levels. In contrast, Arcadia exhibited upregulation of nine genes (AGL14, AGL61, AGL62, MADS1, MADS16, MADS27, MADS33, MADS50, and MADS51), whereas Jenny displayed higher expression of AGL66, MADS16, and MADS56. These variety-specific expression patterns provide preliminary evidence that MADS-box genes may be differentially regulated in cultivars with contrasting stress tolerance phenotypes.
Expression levels of MADS genes in Kentucky Bluegrass treated with drought and salt stresses
According to the previous comparative analysis of tolerance to stress among three Kentucky bluegrass varieties, Arcadia was identified as the primary research subject. Previous studies have indicated that MADS-box genes play important roles in plant development, growth, and responses to various abiotic and biotic stresses (Wang et al., 2018). To examine the MADS-box relative expression levels in the stems and leaves of Kentucky bluegrass (Arcadia) by qRT-PCR, abiotic stresses (drought and salt) were applied. We examined relative expression of sixteen genes (AGL14, AGL28, AGL61, AGL62, AGL66, MADS1, MADS16, MADS26, MADS27, MADS33, MADS34, MADS50, MADS51, MADS56, MADS57, and MADS58) after 0, 4, 6 and 16 h of drought treatment and seven days after salt treatment with different concentration of 0, 100, 150, and 200 mM. Focusing on key MADS-box genes, we found that in leaves under drought, three genes (MADS1, MADS26, and MADS33) showed an initial upregulation followed by a downregulation with the increase in drought stress duration, while the expression level of AGL14 increased with time. In addition, it is worth noting that the expression levels of MADS26, and MADS33 were the highest at 2 h of drought treatment and MADS1 peaked at 6 h. In contrast, the expression level of AGL14 was highest at 16 h (Fig. 6). However, in stems all four genes (AGL14, MADS1, MADS26, and MADS33) exhibited a similar expression pattern, characterized by an initial increase in expression followed by a decrease as the duration of drought stress increased, and the highest expression levels were observed at 2 h (Fig. 7).
Real-time PCR analysis of key representative MADS-box genes in the leaves under drought stress.Expression levels of Kentucky bluegrass (Arcadia) MADS-box genes in leaves under drought stress based on qRT-PCR analyses. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation.
Real-time PCR analysis of key representative MADS-box genes in the stems under drought stress.Expression levels of Kentucky bluegrass (Arcadia) MADS-box genes in stems under drought stress based on qRT-PCR analyses. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation.
Under salt treatment in leaves, a total of five genes (AGL14, AGL66, MADS16, MADS26, and MADS33) showed a similar expression level, characterized by an initial increase followed by a decrease with increasing salt concentration. In contrast, the expression level of MADS51 was significantly upregulated with increasing salt concentration. Notably, the highest expression levels of AGL14, MADS16, and MADS66 were observed at 100 mM. Notably, the highest expression level of MADS51 appeared at 200 mM (Fig. 8). In stems, all genes (AGL14, AGL66, MADS16, MADS26, MADS33, and MADS51) showed a similar expression pattern, characterized by an initial upregulation followed by downregulation, and the highest expression levels of AGL66, MADS16, MADS26, and MADS51 were observed at 100 mM. However, the highest expression levels of AGL14 and MADS33 were observed at 150 mM (Fig. 9).
Real-time PCR analysis of key representative MADS-box genes in the leaves under salt stress.Expression levels of Kentucky bluegrass (Arcadia) MADS-box genes in leaves under salt stress based on qRT-PCR analyses. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation.
Real-time PCR analysis of key representative MADS-box genes in the stems under salt stress.Expression levels of Kentucky bluegrass (Arcadia) MADS-box genes in stems under salt stress based on qRT-PCR analyses. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation.
Some MADS-box genes, such as AGL61 (Fig. S3), AGL62 and MADS34 (Fig. S4), MADS34 and MADS56 (Fig. S5), MADS58 (Fig. S6) and MADS33 (Fig. 9), showed transient expression at specific time points. This temporal expression pattern suggests that this gene may play regulatory roles in the early or intermediate stress response rather than maintaining prolonged activation. The rapid increase at different hours could indicate that these genes functions as an early signal transducer or transcriptional activator, initiating downstream protective or adaptive responses.
All examined genes showed responses to treatments, and most genes showed different tissue-specific expression. Collectively, these results indicate that members of the MADS-box gene family exhibit variable levels of expression patterns under salt and drought stress, suggesting potential involvement in stress response mechanisms.
Analysis and prediction of cis-acting elements in the promoters of MADS transcription factors
A total of 422 cis-acting elements were identified within the 2,000-bp sequences upstream from the translation starting site of each gene. Each MADS-box promoter has been shown to contain three different kinds of cis-regulatory elements: those linked to biotic and abiotic stress, phytohormone response, and plant development and growth (Fig. 10A). Most Kentucky bluegrass MADS-box genes comprise cis-acting elements that regulate plant development and growth, plant hormone and stress responsiveness. The largest percentage, 40.6%, was composed of light-responsive elements. Further, numerous cis-acting elements in MADS promoters are connected to MeJA response (13.1%), ABA (11.3%), anaerobic processes (6.1%), and meristems (5.8%). Some elements associated with drought, low-temperature response, salicylic acid (SA), gibberellin (GA), auxin, defense and stress responsiveness, circadian control, and a few elements related to cell cycle, seed-specific regulation, and wound responsiveness were found in MADS promoters (Fig. 10B). These findings showed that the defense response to different stresses may involve MADS-box genes. However, further studies are needed to elucidate the extent and mechanisms of their involvement fully.
Cis-acting regulatory elements in Kentucky bluegrass MADS-box gene promoters.(A) Cis-acting elements distribution in the promoter of each MADS-box gene in Kentucky bluegrass. (B) Bar charts are used to visually illustrate the different functional cis-acting elements.
Discussion
Cuticular waxes perform an important function for plants to be able to withstand environmental conditions (Singh, Das & Geeta, 2018). It has previously been reported that several plant species exhibit wax deposition on their surfaces in response to salt and drought stress (Sun et al., 2015; Xue et al., 2017). For instance, sesame plants subjected to water shortage have a high concentration of waxes per unit leaf area (Kettani, El Fechtali & Nabloussi, 2024). In our study, scanning electron microscopy (SEM) images showed extensive modifications of epidermal features in different bluegrass varieties. Our findings showed the effective difference in the distribution of wax density, number of papillae, and structure of stomata among the three varieties. Arcadia was observed with more wax density on the upper leaf epidermis and also grew relatively well under drought and salt stress, suggesting a potentially greater ability of this variety to mitigate abiotic stresses. This result was consistent with that of previous findings, which consider cuticular waxes as a barrier to high salinity and drought (Lee & Suh, 2015). Arcadia’s stomata not only had ideal size and density but were also widely distributed, which may help in maintaining water balance against abiotic stress; this finding overlaps with (Chen et al., 2017; Pornsiriwong et al., 2017). Furthermore, Arcadia was observed to have more papillae among all three varieties, which may play a role in regulating water loss and may provide a protective role against abiotic stresses. These results overlap with (Zhu et al., 2023). While these epidermal features may be involved in abiotic stress responses, further research is needed to clarify their specific roles.
MADS TFs are known to play important roles in plant development and have been implicated in responses to various abiotic stresses (Castelán-Muñoz et al., 2019; Zhang et al., 2024). Several MADS-box TFs have been identified in many plants that exhibit stress-responsive characteristics (Guo et al., 2016), but research on MADS-box in Kentucky bluegrass is limited. Therefore, exploring the potential of MADS-box genes could provide valuable insights for improving grass performance under abiotic stress. In the present study, the Kentucky bluegrass variety (Arcadia) was subjected to drought and salt stresses. We identified 16 MADS-box genes using transcriptome sequencing. Our expression data are consistent with previous studies that have reported stress-related functions of MADS-box TFs, such as MADS26 and MADS27 in rice (Arora et al., 2007; Khong et al., 2015; Saha et al., 2015), MADS1 and AGL62 in alfalfa (Dong et al., 2021), MADS34 and 50 in peanut (Mou et al., 2022). For example, BrMADS14 in Brassica rapa responds to salt and drought stresses and was differentially expressed (Saha et al., 2015), which aligns with our observations. In our study, AGL14, MADS1, MADS26, and MADS33 were consistently upregulated under drought stress across all tissues, while under salt stress, AGL14, AGL66, MADS16, MADS26, MADS33, and MADS51 exhibited uniform upregulation across those same tissues. In contrast, the expression of the remaining genes was more tissue-specific (Figs. 6–9). A study in sheep grass also reported tissue-specific expression of MADS-box genes (Wang et al., 2018). Our findings exhibit upregulation of MADS26 under drought stress (Figs. 6, 7), which aligns with the previous findings showing MADS26 upregulation in a wheat genotype that is resistant to drought, suggesting its function in drought stress (Krugman et al., 2011). A study on barley showed that MADS27 exhibited upregulation under salt treatment (Kuang et al., 2019). However, our findings showed that this gene was upregulated in stems when treated with salt (Fig. S6). According to Zhao et al. (2021), the MADS-box gene SiMADS51 in foxtail millet (Setaria italica) is essential for drought stress response, which is consistent with the expression pattern observed in our study. Similarly, OsMADS57 has been shown to positively regulate drought tolerance in Arabidopsis thaliana and rice (Wu et al., 2021). On the other hand, we observed that MADS57 expression was induced by drought stress (Fig. S4). The findings of the abiotic stress treatments in our study were consistent with the previously cited studies and trials. Overall, our expression profiling suggests that MADS-box genes may be involved in stress responses in Kentucky bluegrass, offering a basis for future functional validation and potential applications in stress-resilient breeding strategies. To directly correlate the observed expression patterns with known stress pathways, subsequent research will involve quantitative polymerase chain reaction (qPCR) analysis of canonical drought and salt stress marker genes. This will be crucial to calibrate the transcriptional responses identified in this work and firmly establish their functional significance.
It has been demonstrated that most MADS-box genes respond to stress. According to other findings, we found that biotic and abiotic factors (such as drought, cold, anoxic conditions, zein metabolism, and wounding, etc.) were related to several cis-acting regulatory elements in gene promoters and hormones (abscisic acid (ABA), methyl jasmonate (MeJA), SA, auxin, GA, etc.). The most abundant elements were estimated to be abscisic acid-responsive elements (ABREs), followed by GT1-motif elements. These findings were not completely aligned with the study of alfalfa gene family analysis and MADS-box genes in Eudicots (Dong et al., 2021; Liu et al., 2018). The main difference is that there are fewer ABREs than GT1-motifs. Research has shown that the expression of stress-related genes in plants is controlled by ABREs (cis-acting elements), which are closely related to abiotic stress responsiveness (Dar et al., 2017). Our in-silico findings suggest potential roles of MADS-box genes in responses to various stresses, aligning with previous studies that experimentally demonstrated the significance of these genes under stress conditions (Dong et al., 2021; Duan et al., 2015; Ma et al., 2017). However, these computational predications need further experimental validation. Together, all this analysis showed that the MADS-box gene family is a multipurpose gene family that may mediate abiotic stress in Kentucky bluegrass. More studies on various biological functions of MADS-box genes are needed.
Conclusions
In this study, we analyzed the physiological, morphological, and molecular traits of three Kentucky bluegrass varieties and their response to drought and salt stress. Based on prior evidences linking MADS-box transcription factors to abiotic stress tolerance in monocot species, we particularly focused on MADS-box gene family for systematic investigation. We identified 16 MADS-box genes through transcriptome analysis and characterized their expression factor under stress conditions. Our findings suggest that Arcadia may exhibit relatively greater tolerance to drought and salt stress compared to the other two bluegrass varieties. This potential tolerance appears to be linked to traits such as thicker and more tightly bound cuticular wax, higher chlorophyll content, specific leaf morphology, and stomatal regulation. Quantitative real-time PCR analyses suggest that the key representative genes (AGL14, MADS1, MADS26, and MADS33) under drought stress, and (AGL14, AGL66, MADS16, MADS26, MADS33 and MADS51) under salt stress, were consistently upregulated in all tissues. In silico analysis of MADS-box gene promoters identified cis-acing elements involved in abiotic stresses (salt, drought, heat, etc.), development, and phytohormones (MeJA, ABA, etc.). The expression analysis of MADS-box genes suggests possible involvement in the abiotic stress response pathways of Kentucky bluegrass; while, further functional validation is needed to confirm their regulatory roles. However, this study provides valuable insights that may contribute to understanding the precise functions of MADS-box genes in the future.
Supplemental Information
10.7717/peerj.20933/supp-1Supplemental Information 1Chlorophyl content raw data.
10.7717/peerj.20933/supp-2Supplemental Information 2Go enrichment analysis of transcription factors.
10.7717/peerj.20933/supp-3Supplemental Information 3MIQE checklist.
10.7717/peerj.20933/supp-4Supplemental Information 4Promoters raw data.
10.7717/peerj.20933/supp-5Supplemental Information 5Heatmap raw data.
10.7717/peerj.20933/supp-6Supplemental Information 6SEM raw data.
10.7717/peerj.20933/supp-7Supplemental Information 7qPCR raw data.
10.7717/peerj.20933/supp-8Supplemental Information 8List of primers.
10.7717/peerj.20933/supp-9Supplemental Information 9Drought and salt stress raw photographs.
10.7717/peerj.20933/supp-10Supplemental Information 10SEM raw photos.
10.7717/peerj.20933/supp-11Supplemental Information 11Experimental workflow and summary of major findings in Kentucky bluegrass stress response study.The schematic overview outlines the complete experimental and result pipeline. (A) Greenhouse-grown Kentucky bluegrass varieties (K.B.G., Arcadia, Jenny) were maintained under controlled conditions. (B) Drought and salt stress treatments were applied. (C) Leaves and stems were collected. (D) Physiological and molecular analyses were conducted. Major results (stress responses, microstructure changes, variable expression of MADS-box genes, cis-element patterns) are summarized in panel E and F.
10.7717/peerj.20933/supp-12Supplemental Information 12Differential expression heatmap of MADS-box transcripts in K.B.G., Arcadia, and Jenny varieties.Heatmap of 26 significant MADS-box transcripts (|log₂FC| > 1, q < 0.05) across three Kentucky bluegrass varieties. Colors show normalized log₂ fold changes (red = high, blue = low), with hierarchical clustering revealing distinct expression patterns among varieties.
10.7717/peerj.20933/supp-13Supplemental Information 13Real-time PCR analysis of selected MADS-box genes in the leaves under drought stress.Expression levels of Kentucky bluegrass (Arcadia) MADS-box genes in leaves under drought stress based on qRT-PCR analyses. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation.
10.7717/peerj.20933/supp-14Supplemental Information 14Real-time PCR analysis of selected MADS-box genes in the stems under drought stress.Expression levels of Kentucky bluegrass (Arcadia) MADS-box genes in stems under drought stress based on qRT-PCR analyses. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation.
10.7717/peerj.20933/supp-15Supplemental Information 15Real-time PCR analysis of selected MADS-box genes in the leaves under salt stress.Expression levels of Kentucky bluegrass (Arcadia) MADS-box genes in leaves under salt stress based on qRT-PCR analyses. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation.
10.7717/peerj.20933/supp-16Supplemental Information 16Real-time PCR analysis of selected MADS-box genes in the stems under salt stress.Expression levels of Kentucky bluegrass (Arcadia) MADS-box genes in stems under salt stress based on qRT-PCR analyses. Significant differences (p < 0.05) are indicated by different letters, and error bars indicate ± standard deviation.
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