Background

Cytokinin (CK) is a small molecule plant hormone composed of N6-adenine derivatives, playing a crucial role in plant growth, development, and stress response [1, 2]. CKs commonly found in higher plants include N6-Δ2-isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydro-zeatin (DHZ). In plants, CKs primarily exist in the forms of iP, tZ, and their nucleosides, nucleotides and glycosides [3]. The predominant forms of CKs vary significantly among different plants, tissues within the same plant, and various developmental stages [4].

Numerous studies have indicated a correlation between plant leaf senescence and CK content [4, 5]. Research by Zhang, et al. [6] elucidated the molecular mechanism of CK decline during leaf senescence, demonstrating that plants can regulate leaf senescence by modulating endogenous CK levels.

Isopentenyltransferase (IPT) is a crucial rate-limiting enzyme in CK synthesis, with the IPT gene first identified in Agrobacterium tumefaciens [7]. It can be classified into two classes based on substrate specificity: adenine phosphate-IPT (ATP/ADP-IPT) and transfer RNA-IPT (tRNA-IPT) [8, 9]. Overexpression of the IPT gene results in increased CK content [10]. Transgenic plants overexpressing IPT display varying degrees of CK syndrome, including reduced root growth, altered apical dominance, and changes in stem and leaf growth [11]. This gene not only affects plant growth and development but also responds to abiotic stress. For example, the COR15a promoter-IPT gene enhanced low temperature tolerance in sugarcane (Saccharum officinarum) [12], and regulated expression of the IPT gene in peanut (Arachis hypogaea) improved peanut’s drought torelance [13]. Overexpression of the Agrobacterium tumefaciens IPT gene in cotton (Gossypium hirsutum) improved salt tolerance and delayed senescence of cotton leaves [14]. Although IPT genes in Agrobacterium tumefaciens and plants have been widely studied, comprehensive research on the regulation mechanism of the IPT gene is still lacking.

Our laboratory, Medicago truncatula was treated with 6-benzylaminopurine (6-BA) and lovastatin, and several key regulatory genes potentially involved in CK response were screened using high-throughput sequencing, including the CK synthesis regulatory gene MtIPT. The IPT gene of M. truncatula was cloned using PCR, and the MtIPT gene was found to respond to drought stress.

Creeping bentgrass (Agrostis stolonifera) is a cold-season turf grass known for its ability to grow new roots and stems via stolons. Despite its soft grass color, rapid growth, salt tolerance, cold resistance, and resilience to low mowing and foot traffic, creeping bentgrass exhibits relatively weak drought tolerance [15]. Genetic transformation technology has been well developed for creeping bentgrass with high conversion efficiency. Compared with the wild type (WT), phenotypic changes in transgenic (OE) creeping bentgrass are easily observable, which facilitates the study of gene function. Due to difficulties in seed production and its genetic characteristics of self-incompatibility, traditional breeding methods struggle to genetically improve creeping bentgrass [16]. Therefore, genetic engineering has become a focal point for enhancing creeping bentgrass. It is hoped that superior germplasm resources of creeping bentgrass can be obtained through the introduction and screening of exogenous genes.

The purpose of this study is to explore the regulatory mechanism of the MtIPT gene on the growth and abiotic stress response of creeping bentgrass through gene transformation and overexpression. Building upon previous studies [17, 18], this research investigates various aspects of the MtIPT gene’s regulatory mechanism, such as cell structure and hormone interactions. This study offers a new perspective for comprehensively understanding IPT gene function and CK regulation, laying a foundation for developing new varieties of creeping bentgrass.

Results

The acquisition of transgenic plants

The MtIPT gene (XM_003590629.4), 903 bp, was cloned via PCR (Fig. S1). Subsequently, the MtIPT gene was integrated into the genome of creeping bentgrass through agrobacterium-mediated transformation, resulting in 32 OE plants. The transcription of MtIPT in OE plants was confirmed by qRT-PCR. As depicted in Figure S2, MtIPT was effectively transcribed in creeping bentgrass. Moreover, the expression of the MtIPT gene was significantly higher in the leaf (13.5-fold) and stem (12.3-fold) tissues compared with the root section of transgenic creeping bentgrass (Fig. 1). Therefore, the above-ground portion of transgenic creeping bentgrass was selected as the primary material for subsequent study.

Fig. 1
figure 1

QRT-PCR analysis of the relative expression of the MtIPT in different tissues of transgenic plant. Data represent the average from four biological replicates. Data are shown as means and the error bars indicate the standard deviation (± SD). Compared with root, significant difference was indicated with asterisks (ns, *, **, *** or ****) at ns, P < 0.05, 0.01, 0.001 or 0.0001 by Student’s t-test

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Subcellular localization

The MtIPT gene was ligated into the 3302Y vector containing yellow fluorescent protein gene (YFP) and introduced into tobacco leaves via agrobacterium-mediated transformation. Using confocal laser microscopy, the isopentenyltransferase encoded by MtIPT was observed to localize in the cell membrane and nucleus (Fig. 2).

Fig. 2
figure 2

Subcellular localization of MtIPT protein. 3302Y, control with empty vector. 3302Y-IPT, MtIPT-GFP fusion. Bars = 25 μm

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Phenotype and internal structure characteristics of wild-type and transgenic creeping bentgrass

WT and OE plants matured after 12 weeks of cultivation in soil (Fig. 3A). Compared with WT, OE plants exhibited significantly reduced stem length (26%) (Fig. 3B), shorter roots (40%) (Fig. 3C), and thinner leaves (47%) (Fig. 3D), while displaying a notably higher chlorophyll content (33%) (Fig. 3E). Interestingly, the tips of some leaves appeared red (Fig. 3A). Staining results from cross-sections indicated no discernible difference in leaf cell structure between WT and OE plants (Fig. 3F). However, scanning electron microscopy (SEM) results revealed that the content of wax crystals on the surface of stomata (Fig. 3G) was significantly higher in OE plants compared with WT. This enhancement may assist OE plants in reducing transpiration and retaining more water during periods of drought.

Fig. 3
figure 3

Phenotypic characteristics, microstructure and chlorophyll content of plants. (A) Wild-type (WT) plants and transgenic (OE) plants were grown in soil (Nutrient soil: vermiculite: perlite = 1:1:1) separately and photographed after maturity. Statistical analysis of the differences in stem length (B), root length (C), leaf width (D) and chlorophyll content (E) between representative WT and OE plants. Data represent the average from three biological replicates. Data are means of three biological replicates ± standard deviation (SD). OE lines compared with WT, significant difference was indicated with asterisks (ns, *, **, *** or ****) at ns, P < 0.05, 0.01, 0.001 or 0.0001 by Student’s t-test. (F) Cross sections of leaves of WT and OE plants. G Scanning electron microscope images of stomata of WT and OE leaves

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Response of MtIPT gene to drought stress

Four M. truncatula plants were treated with drought using 20% (w/v) Polyethylene glycol (PEG-6000). The results indicated that the expression of the MtIPT gene initially increased, peaked at 8 h, and then decreased within 24 h. However, compared with the baseline (0 h), the expression of the MtIPT gene showed significant increases at 0.5 h (1.9-fold), 1 h (7.1-fold), 2 h (6.8-fold), 4 h (8.5-fold), 6 h (12.0-fold), 8 h (13.1-fold), 12 h (7.4-fold), and 24 h (4.6-fold) (Fig. 4). This indicates that the MtIPT gene responds to drought stress.

Fig. 4
figure 4

The expression levels of MtIPT gene were analyzed at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h through drought treatment on healthy Medicago truncatula. Data represent the average from four biological replicates. Data are shown as means and the error bars indicate the standard deviation (± SD). Compared with 0 h, significant difference was indicated with asterisks (ns, *, **, *** or ****) at ns, P < 0.05, 0.01, 0.001 or 0.0001 by Student’s t-test

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MtIPT gene enhances drought resistance of creeping bentgrass by increasing antioxidant enzymes

WT and OE plants were cultured under identical conditions, and plants in similar physiological states were selected for drought treatment (Fig. 5A). By the 14th day of drought treatment, the leaves of WT plants had significantly yellowed and some had withered, whereas the OE plants remained green (Fig. 5B). By the 22nd day, WT plants had experienced extensive leaf death, whereas the transgenic plants only showed partial wilting, with most leaves remaining green (Fig. 5C).

The contents of chlorophyll, antioxidant enzymes, and soluble sugars in the leaves of WT and OE plants were measured after 0 and 14 days of natural drought treatment. Following drought treatment, compared with WT plants, the peroxidase (POD) (77%), superoxide dismutase (SOD) (16%), and ascorbate peroxidase (APX) (28%) contents of OE plants significantly increased (Fig. 5D-F), while the malondialdehyde (MDA) content noticeably decreased (8%) (Fig. 5G). Additionally, the soluble sugar content increased significantly (17%) (Fig. 5H). Furthermore, the chlorophyll content in the leaves of OE plants was significantly higher (47%) than that in WT plants after drought treatment (Fig. 5I). These results indicate that overexpression of the MtIPT gene enhances the drought torelance of creeping bentgrass by influencing its antioxidant capacity, photosynthetic performance, and osmotic regulation.

Fig. 5
figure 5

Drought treatment of wild type (WT) and transgenic (OE) plants. Comparison of morphology of WT and OE plants under drought treatment at day 0 (A), day14 (B), day 22 (C). Physiological indexes of WT and OE plants were measured before (day 0) and after (day 14) drought treatment, including peroxidase (POD) (D), super oxide dimutese (SOD) (E), ascorbate peroxidase (APX) (F), malondialdehyde (MDA) (G), soluble sugar (H) and chlorophyll content (I). Data represent the average from three biological replicates. Data are shown as means and the error bars indicate the standard deviation (± SD). OE lines compared with WT, significant difference was indicated with asterisks (ns, *, **, *** or ****) at ns, P < 0.05, 0.01, 0.001 or 0.0001 by Student’s t-test

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Transgenic plants improve shading by reducing the degradation of chlorophyll in leaves

The leaves of WT and OE plants were placed in 3 mM 2-Morpholinoethanesulphonic acid (MES) solution for dark culture (Fig. 6A). After 5 days, the leaves of WT plants showed slight yellowing, while those of OE plants exhibited minimal change (Fig. 6B). By day 8, the leaves of WT plants had almost completely yellowed, whereas those of OE plants only showed partial yellowing (Fig. 6C). Chlorophyll determination results indicated that the chlorophyll content in OE plants was significantly higher (83% and 63%) than in WT plants on days 5 and 8 (Fig. 6D). This demonstrates that the MtIPT gene can enhance plant shade tolerance by reducing chlorophyll degradation and delaying leaf senescence.

Fig. 6
figure 6

Shade treatment of wild type (WT) and transgenic (OE) plants. WT, OE2 and OE16 leaves with the same growth state were selected from three plants respectively as three biological repeat samples. The leaves of WT and OE plants were darkened in 3 mM 2-Morpholinoethanesulphonic acid (MES) solution, and the state of the leaves was recorded by photos on day 0 (A), day 5 (B) and day 8 (C). D Chlorophyll content of WT and OE plants before and after shading treatment. Data represent the average from three biological replicates. Data are shown as means and the error bars indicate the standard deviation (± SD). OE lines compared with WT, significant difference was indicated with asterisks (ns, *, **, *** or ****) at ns, P < 0.05, 0.01, 0.001 or 0.0001 by Student’s t-test

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Hormonal changes of transgenic creeping bentgrass

The hormone determination results revealed that overexpression of the MtIPT gene in creeping bentgrass led to an increase in CK trans-zeatin-riboside (88%) (Fig. 7D) and isopentenyl adenosine (35%) (Fig. 7F) content. Conversely, the CK DL-dihydrozeatin riboside content decreased (13%) (Fig. 7E), indicating that the MtIPT gene primarily catalyzed the synthesis of isopentenyl adenosine and trans-zeatin-riboside. Additionally, besides the significant changes in CK content, overexpression of MtIPT notably increased the levels of auxin (26%) (Fig. 7A), brassinosteroid (59%) (Fig. 7G), and abscisic acid (8%) (Fig. 7B) in OE plants. It also reduced the content of the hormone methyl jasmonate (34%) (Fig. 7H). These results suggest that various hormones co-regulate the changes in growth, development, and abiotic stress response in OE plants. Compared with WT plants, the GA3 content in OE plants remained almost unchanged (Fig. 7C).

Fig. 7
figure 7

Hormone content in the ground parts of wild type (WT) and transgenic (OE) plants. A Auxin (IAA) content. B Abscisic acid (ABA) content. C Gibberellin 3 (GA3) content. D Cytokinin trans-Zeatin-riboside (ZR) content. E Cytokinin DL-dihydrozeatin riboside (DHZR) content. F Cytokinin isopentenyl adenosine (IPA) content. G Brassinosteroid (BR) content. H methyl jasmonate (MeJA) content. The unit is ng/g· FW. Data represent the average from three biological replicates. Data are shown as means and the error bars indicate the standard deviation (± SD). OE lines compared with WT, significant difference was indicated with asterisks (ns, *, **, *** or ****) at ns, P < 0.05, 0.01, 0.001 or 0.0001 by Student’s t-test

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Epbrassinolide promotes the expression of MtIPT gene

According to the results of hormone determination, we found that overexpression of the MtIPT gene resulted in a significant increase in brassinosteroid content (Fig. 7G). It is intriguing to explore whether this increase in brassinosteroid content could also enhance the expression of MtIPT. Therefore, 24-epibrassinolide (EBR) was used for exogenous spraying. After EBR treatment, MtIPT gene expression was measured at different time points. As depicted in Fig. 8, compared with 0 h, MtIPT gene expression was up-regulated at 0.5 h (1.1-fold), 1 h (1.7-fold), 6 h (1.4-fold), 9 h (1.1-fold), 12 h (1.5-fold), and 24 h (2.7-fold), illustrating that the elevated concentration of exogenous EBR also contributed to improving the expression level of the MtIPT gene. Consequently, it is suggested that there is a certain synergistic effect between cytokinin and brassinosteroid, and they participate in regulating plant life processes through interaction.

Fig. 8
figure 8

The expression levels of MtIPT gene were analyzed at 0 h, 0.5 h, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h after exogenous application of 0.1 µmol 24-epbrassinolide (EBR) on healthy Medicago truncatula. Data represent the average from four biological replicates. Data are shown as means and the error bars indicate the standard deviation (± SD). Compared with 0 h, significant difference was indicated with asterisks (ns, *, **, *** or ****) at ns, P < 0.05, 0.01, 0.001 or 0.0001 by Student’s t-test

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Results of transcriptome sequencing

High-throughput sequencing was conducted to investigate changes in growth, development, and abiotic stress response in OE plants. A total of 39.33 Gb of clean data were obtained, with all samples yielding 6.37Gb of clean data and a Q30 base percentage of 93.57% or higher (Table 1). A total of 72,209 unigenes were assembled, with an N50 of 1,697. Additionally, 36,452 unigenes were annotated following comparison with multiple databases. Differentially expressed genes (DEGs) were identified based on their expression levels across different samples. In transgenic creeping bentgrass compared with WT, 8,700 DEGs were identified, with 4,122 up-regulated and 4,578 down-regulated genes.

Table 1 Data quality of RNA-Seq in wild-type and transgenic creeping bentgrass
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Based on GO analysis, the main enriched biological processes of up-regulated DEGs include triterpenoid biosynthetic process, glycogen biosynthetic process, brassinosteroid metabolic process, inositol biosynthetic process, neutral lipid biosynthetic process, inositol phosphate-mediated signaling, oxidation-reduction process, cytokinin metabolic process, brassinosteroid homeostasis, steroid biosynthetic process, among others. The main enriched biological processes of down-regulated DEGs include cell surface receptor signaling pathway, DNA integration, plant-type secondary cell wall biogenesis, mitochondrial calcium ion transmembrane transport, RNA-dependent DNA biosynthetic process, regulation of mitotic spindle organization, sterol metabolic process, de novo protein folding, regulation of intracellular signal transduction, simple leaf morphogenesis, among others (Fig. 9A) (Tables S1, S2, S3). These processes may be closely related to the slower growth observed in OE plants.

The main enriched cellular components of up-regulated DEGs include lipid droplet, obsolete axoneme part, integral component of chloroplast outer membrane, chloroplast stroma, extrinsic component of mitochondrial inner membrane, and peptidase complex, among others. Conversely, the main enriched cellular components of down-regulated DEGs include apoplast, extracellular region, spindle, cell wall, spindle microtubule, microtubule, and integral component of plasma membrane, among others (Fig. 9B) (Tables S4, S5, S6).

The main enriched molecular functions of up-regulated DEGs include oxidoreductase activity, lanosterol synthase activity, transaminase activity, 1,4-alpha-glucan branching enzyme activity, phosphoric diester hydrolase activity, and cytokinin dehydrogenase activity, among others. On the other hand, the main enriched molecular functions of down-regulated DEGs include ADP binding, protein kinase activity, polysaccharide binding, threonine synthase activity, monooxygenase activity, and DNA-binding transcription factor activity, among others (Fig. 9C) (Tables S7, S8, S9).

KEGG analysis revealed that DEGs were predominantly involved in pathways such as terpenoid biosynthesis, starch and sucrose metabolism, phenylpropanoid biosynthesis, linoleic acid metabolism, flavonoid biosynthesis, tryptophan metabolism, inositol phosphate metabolism, plant hormone signal transduction, MAPK signaling pathway-plant, steroid biosynthesis, betalain biosynthesis, photosynthesis, anthocyanin biosynthesis, brassinosteroid biosynthesis, cutin, suberine and wax biosynthesis, carotenoid biosynthesis, and zeatin biosynthesis, among others (Fig. 9D).

Fig. 9
figure 9

Global analysis of gene expression between wild type (WT) and transgenic (OE) plants. A The biological process analysis of Gene Ontology (GO) enrichment of the all annotated differentially expressed genes (DEGs) in OE-vs-WT group. B The cellular component analysis of GO enrichment of the all annotated DEGs in OE-vs-WT group. C The molecular function analysis of GO enrichment of the all annotated DEGs in OE-vs-WT group. Note: OE-vs-WT: transgenic plants verse wild type plants. 20 most significant GO categories were selected according to the pvalue. D Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the annotated DEGs in OE-vs-WT group. Note: OE-vs-WT: transgenic plants verse wild type plants. 20 most significant KEGG pathways were selected according to the qvalue. Note: GeneRatio: Ratio of the number of genes associated with the category/pathway among the genes used for enrichment analysis to the total number of genes used for enrichment analysis. A higher ratio indicates that more DEGs are enriched in this category/pathway. Enrichment factor: A parameter that reflects gene enrichment, indicating the ratio of the proportion of genes annotated to an category/pathway in DEGs to the proportion of genes annotated to that category/pathway in all genes. A larger enrichment factor indicates that the enrichment level of DEGs in this category/pathway is more significant. Pvalue: Statistically significant levels for enrichment analysis. qvalue is the pvalue after correction for multiple hypothesis testing. The smaller qvalue indicates that the enrichment significance of DEGs in this category/pathway is more reliable. The size of the circle indicates the number of genes enriched in the category/pathway

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Table 2 Annotation of selected genes associated with stress tolerance
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The phenotypes observed in OE plants prompted us to focus on DEGs associated with plant growth and stress response to elucidate the molecular mechanisms of MtIPT-mediated alterations in plant development and stress resistance. Given the slow growth phenotype observed in aboveground parts and roots of OE plants, biological pathways related to auxin and roots were analyzed in OE plants, including microtubule-associated proteins (MAPs), auxin response factors (ARFs), indoleacetic acid-induced proteins (IAAs), SMALL AUXIN UP RNA (SAUR), auxin transporter-like proteins (AUX1), and IAA-amino acid hydrolase ILR1-like proteins (ILR1). These genes showed both up-regulation (10 unigenes) and down-regulation (14 unigenes) in OE plants. Simultaneously, a significant number of stress-related candidate genes were identified (Table 2). Additionally, 8 functional genes were selected for verification of their expression levels, which were consistent with the expression profiles observed in the RNA-seq data (Fig. 10A-H).

Fig. 10
figure 10

Relative expression of the selected DEGs (A-H) determined by qRT-PCR. Data represent the average from four biological replicates. Data are shown as means and the error bars indicate the standard deviation (± SD). Significant differences between the two samples were indicated with asterisks (ns, *, **, *** or ****) at ns, P < 0.05, 0.01, 0.001 or 0.0001 by Student’s t-test

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Discussion

IPT is a key rate-limiting enzyme in CK synthesis, and numerous studies have demonstrated its close association with plant growth and development. Sa, et al. [19] found that overexpression of the IPT gene altered endogenous CK levels, affecting plant morphology, floral organ systems, and reproductive processes. Similarly, transgenic GmIPT tobacco (Nicotiana tabacum) showed inhibited root and aboveground growth, shortened internodes, thicker phenotypes, and darker green leaves [20]. In this study, overexpression of MtIPT in creeping bentgrass led to slower growth in aboveground parts and roots, with darker leaf color, consistent with findings by Ye, et al. [20]. GO analysis from high-throughput sequencing revealed that down-regulated DEGs were enriched in biological processes such as DNA integration, plant-type secondary cell wall biogenesis, mitochondrial calcium ion transmembrane transport, RNA-dependent DNA biosynthesis, regulation of mitotic spindle organization, protein folding, and regulation of intracellular signal transduction. These findings suggest that MtIPT may retard plant growth by influencing DNA replication and protein synthesis to some extent.

The MtIPT gene not only influences growth and development but also regulates plant response to abiotic stress. Various studies have shown that IPT genes in plants respond to stresses like high temperature [21], low temperature [12], drought [17], and salt [22]. Through high-throughput sequencing, Xu, et al. [23] found differential expression of receptor kinase, stress defense, protein modularization, and protein degradation genes between transgenic IPT and wild-type plants under drought stress. In this study, overexpression of MtIPT enhanced drought tolerance and shade tolerance in creeping bentgrass by delaying leaf senescence. High-throughput sequencing also highlighted DEGs related to plant hormone signal transduction, steroid biosynthesis, photosynthesis, flavonoid biosynthesis, carotenoid biosynthesis, anthocyanin biosynthesis, oxidation-reduction process, cytokinin metabolism, and wax biosynthesis in OE plants compared with WT.

Drought can increase the levels of O− and H2O2 levels in plant tissues, inducing rapid accumulation of reactive oxygen species (ROS) and oxidative damage to proteins and lipids [24, 25]. Plants have developed defense mechanisms to regulate ROS levels, including antioxidant enzymes like POD, SOD [26], and non-enzymatic antioxidants such as flavonoids [27, 28]. In this study, OE plants showed significantly up-regulated expression of antioxidase genes (APX2, PEX11, and PER1), higher levels of antioxidant enzymes (POD, SOD, APX), lower levels of malondialdehyde (MDA), and increased expression of flavonoid synthesis genes (F3H, C12RT1, DFR) compared with WT. These results indicate that the MtIPT gene enhances drought tolerance in OE plants by improving antioxidant capacity.

Anthocyanins are also a kind of flavonoids, which act as effective and broad-spectrum protective compounds and can accumulate in vacuoles, functioning as osmotic regulators to improve plant drought tolerance [29]. Nogues, et al. [30] found that UV-B-induced anthocyanins reduced pea (Pisum sativum) damage under drought stress, enhancing drought tolerance. The BZ1 gene, encoding anthocyanidin 3-O-glucosyltransferase, is responsible for anthocyanin synthesis [31]. ANS is a key enzyme at the end of plant anthocyanin biosynthesis pathway [32]. In this study, BZ1 and ANS genes were significantly up-regulated in OE plants. This suggests that overexpression of the MtIPT gene increases anthocyanin content by promoting the up-regulation of BZ1 and ANS, thereby reducing drought-induced damage to OE plants.

Epidermal wax forms a hydrophobic layer that coats the plant surface, crucial for maintaining water balance and preventing water loss [33, 34]. In this study, we observed that the MtIPT gene significantly increased the expression of the wax synthesis gene MAH1 (Fig. 10D) and influenced the content and morphology of wax crystals around stomata (Fig. 3G) in OE plant leaves. This finding helps to reduce water loss and may be a key factor contributing to increased drought resistance in OE plants.

CRE1, AHK2, and AHK3, are CK signal transduction genes encoding arabidopsis histidine kinase that act as negative regulators in ABA signal transduction. Mutants cer1, ahk2, and ahk3 in Arabidopsis exhibit enhanced drought tolerance [35]. CK signal transduction B-type ARR protein mediates drought response in Arabidopsis as a negative regulator, and the improved drought tolerance of arr mutant plants is associated with enhanced cell membrane integrity, increased anthocyanin biosynthesis, abscisic acid hypersensitivity, and decreased stomatal pore size [36]. In this study, compared with WT plants, the expression levels of CER1, AHK2, and AHK3 genes were down-regulated, along with down-regulation of four B-type ARR genes and up-regulation of one in the CK signaling pathway of OE plants. This modulation may contribute significantly to the enhanced drought tolerance observed in OE plants.

Senescence-associated genes (SAGs) are pivotal in regulating leaf senescence. Cytochrome P450 CYP89A9 participates in the formation of major chlorophyll catabolites during leaf senescence in plants [37]. Jing, et al. [38] demonstrated that melatonin suppressed age- and dark-induced leaf senescence in cucumber (Cucumis sativus) by reducing the expression of SAG20 and the cell-death-related gene PDCD, while increasing antioxidant capacity and chlorophyll biosynthesis. In this study, compared with WT plants, the expression of SAG20 (Fig. 10F) and cytochrome P450 CYP89A9 (Fig. 10G) genes in OE plants were significantly decreased. Concurrently, genes related to photosynthesis (ATPF1A, PSBQ, PETF) and antioxidase-related genes were notably up-regulated in OE plants. Therefore, overexpression of the MtIPT gene likely delays leaf senescence by enhancing chlorophyll content, photosynthetic capacity, and antioxidant capacity in OE plants.

Plant hormones play critical roles in growth, development, and abiotic stress response through found complex interactions. Xia, et al. [39] demonstrated that brassinosteroids in tomato (Solanum lycopersicum) influence apical dominance by integrating signals from auxin, abscisic acid, gibberellin, and cytokinin, along with sugar signaling. Li, et al. [40] revealed that auxin, brassinosteroid, and cytokinin interact to regulate root meristem development, involving auxin signaling components like PIN7 and SHY2. In this study, in addition to significant increases in isopentenyl adenosine and trans-zeatin-riboside, the levels of auxin and brassinosteroid were significantly elevated in MtIPT-overexpressed plants. Moreover, the expression of the MtIPT gene increased notably upon exogenous EBR spraying, indicating that CKs influence auxin and brassinosteroid synthesis by regulating gene expression. Conversely, brassinosteroids can also affect CK synthesis, but further mechanistic studies are needed.

Brassinosteroids play crucial roles in plant response to abiotic stress. Chen, et al. [41] demonstrated that the brassinosteroid-deficient cotton mutant pag1 (pagoda1) exhibited increased sensitivity to drought stress compared with wild-type plants. Chen, et al. [42] indicated that brassinosteroid enhanced the heat tolerance of plants by inhibiting ERF49 expression through BZR1. Additionally, Liu, et al. [43] confirmed that brassinosteroid signal transduction plays a vital role in regulating plant salt tolerance through a series of Arabidopsis brassinosteroid mutant and overexpressed materials. In this study, changes in hormone levels strongly suggest that brassinosteroid variation is a critical aspect of MtIPT gene regulation in transgenic creeping bentgrass under abiotic stress conditions.

Fig. 11
figure 11

A proposed working model of MtIPT in regulating plant growth, drought stress response and leaf senescence in Agrostis stolonifera. The dashed blue arrow indicates the corresponding relationship, the black up arrow indicates the up-regulating, and the black down arrow indicates the down-regulating

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Conclusion

In conclusion, we have proposed a working model outlining the molecular mechanisms by which MtIPT regulates plant growth, drought stress response, and leaf senescence (Fig. 11). The IPT gene from M. truncatula was overexpressed in creeping bentgrass, resulting in observed slower growth of both aboveground and root parts, thinner leaves, and darker leaf color compared with WT plants. Physiological indices measured after drought and shade stress treatments demonstrated significantly enhanced drought resistance and improved shade tolerance in OE plants, achieved through delayed leaf senescence. The wax content in the leaf epidermis, critical for water retention, was found to be closely linked to these traits. Scanning electron microscope results revealed a notable increase in wax crystal content around the stomata of OE plants, contributing to reduced water loss under drought stress conditions. High-throughput sequencing uncovered numerous DEGs associated with growth, development, and response to abiotic stress, including those related to auxin signaling, root development, photosynthesis, and peroxisome function, among others. Importantly, the overexpression of the MtIPT gene not only increased the levels of cytokinins (isopentenyl adenosine and trans-zeatin-riboside) in OE plants but also significantly elevated auxin and brassinosteroid levels. These findings suggest that MtIPT influences the physiological changes in OE plants by regulating the interactions among various hormones. These results provide valuable insights and a foundation for further exploration into the molecular mechanisms underlying the effects of the IPT gene on plant growth, development, and response to abiotic stresses. Additionally, they offer new avenues for genetic improvement of creeping bentgrass.

Material and method

MtIPT gene cloning, plasmid construction and plant transformation.

The material used in this study included M. truncatula R108 seeds obtained from the Noble Foundation in the United States. These seeds were planted in soil (vermiculite: nutrient soil: perlite = 1:1:1) and cultivated for 2 months in a greenhouse to mature plants. Total RNA was extracted from M. truncatula leaves, and the MtIPT gene (XM_003590629.4), 903 bp, was cloned using PCR. Subsequently, the gene was ligated into the 3302-UBI vector containing the CaMV35S promoter to construct a plant expression vector. This vector was then introduced into the genome of the WT creeping bentgrass ‘Penn A4’ using the agrobacterium-mediated method, resulting in the generation of OE plants.

Subcellular localization of MtIPT protein.

Tobacco seeds were germinated under conditions similar to those used for M. truncatula. The MtIPT gene was cloned into the 3302Y vector, which contains the YFP gene, and subsequently transformed into Agrobacterium EHA105. The properties that YFP can be expressed in various subcellular structures can be used to study the localization of genes. When the tobacco plants reached 5–6 weeks of age, both the 3302Y vector and 3302Y-MtIPT construct were separately introduced into tobacco leaves using agrobacterium-mediated transformation. The reason for using 3302Y vector containing the YFP gene as a control is to distinguish between plant autofluorescence and YFP fluorescence to avoid spurious interference. After a two-day period of dark culture, subcellular localization was observed under the YFP channel of a confocal microscope. In order to facilitate observation, the fluorescence color was set to green in confocal microscopy.

Response of MtIPT gene to drought stress.

Four mature M. truncatula plants were carefully selected and subjected to drought treatment using 20% (w/v) PEG 6000, following the method described by [44, 45] with modifications. Samples were collected at intervals of 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h. At each time point, 0.1 g of M. truncatula leaves was rapidly frozen with liquid nitrogen and stored at -80 °C. Total RNA was extracted from the samples using the Trizol method and reverse-transcribed into cDNA for subsequent qRT-PCR analysis.

Plant materials, propagation and abiotic stress treatment.

WT and OE plants overexpressing MtIPT were transplanted from the growth medium into 15 cm × 15 cm pots filled with soil (vermiculite: nutrient soil: perlite = 1:1:1) and placed under a 16 h/8 h light/dark cycle at 23 °C. After 12 weeks of growth, WT and OE plants were carefully removed from the pots and cleaned thoroughly. Observations were made on the growth of aboveground parts and roots, and measurements were taken for stem length, root length, leaf width, and chlorophyll content of the plants. Samples from WT, OE2, and OE16 plants were selected for measurement of these parameters, each with three biological replicates.

For the drought stress experiments, WT control and OE plants were planted in pots containing 200 g of soil, following the initial maintenance procedure described above. The soil field moisture capacity of each pot was measured according to the method described by Yuan [46] with modifications. After 12 weeks of growth, plants at similar developmental stages were chosen for drought treatment. Natural drought stress conditions were applied to ensure the authenticity and accuracy of physiological parameters. By the 14th day of drought treatment, the soil water content was calculated on the basis of soil field moisture capacity [47]. The water content of the soil planted with WT plants was measured as 34%, 32%, and 33% of field moisture capacity, while that of soil planted with OE2 plants was 33%, 32%, and 33% of field moisture capacity, and OE16 plants showed 34%, 33%, and 33% of field moisture capacity, respectively. Observations and photographs were taken daily to monitor leaf changes, and leaf samples were collected on days 0 and 14 of the treatment. These samples were rapidly frozen with liquid nitrogen and stored at -80 °C for subsequent analysis of chlorophyll content, soluble sugar content, MDA content, and antioxidant enzyme activities including POD, SOD, and APX. WT, OE2, OE7, and OE16 plants were selected as samples for measuring physiological parameters, each with three biological replicates.

Regarding the shade treatment, leaves of WT and OE plants under the same soil culture conditions mentioned above were cultured in vitro in 3 mM MES solution under dark conditions. MES is a zwitterionic buffer that mimics the plant’s natural environment. Our laboratory previously determined through ion concentration gradient experiments that 3 mM MES is effective for the survival of creeping bentgrass. MES solution was used to simulate an in vitro leaf culture environment, and the plants were subjected to a light shading experiment. This method was chosen for its simplicity and ease of observation of experimental results. Daily observations and photographs were taken to monitor leaf changes, and chlorophyll content was measured on days 0, 5, and 8 of the shading treatment. WT, OE2, OE16 leaves with the same growth state were selected from the three plants respectively as three biological repeat samples.

Tissue sections and scanning electron microscopy.

Paraffin transverse sections of creeping bentgrass leaves were prepared according to the method described by Yuan, et al. [48] with modifications. These sections were then stained with ferro red and green fixation to observe the cellular morphological structure of creeping bentgrass leaves under a light microscope (NIKON, Japan). Samples from WT, OE2, and OE16 plants were selected for sectioning, with each sample having three biological replicates.

Fresh plant leaves were fixed using an electron microscope fixative solution (Servicebiop, China), followed by post-fixation, dehydration, drying, and conductive treatment. The epidermal structure of the plant leaves was subsequently observed, and images were captured using SEM (HITACHI, China) [49]. Samples from WT, OE2, and OE16 plants were chosen for SEM imaging, each with three biological replicates.

Determination of chlorophyll content, soluble sugar content, antioxidant enzyme content and malondialdehyde content.

Chlorophyll content was measured following established protocols [50]. The activities of antioxidant enzymes, including POD, SOD and APX, were determined according to Nahakpam, et al. [51]. Malondialdehyde content was quantified using the method described by Esterbauer, et al. [52]. Plant soluble sugar content was assessed using the Plant Soluble Sugar Content Assay Kit following the manufacturer’s instructions.

Hormone Contents Assay.

The leaves of 12-week old WT and OE plants were collected and crushed with liquid nitrogen immediately after rapid freezing. Hormone contents were measured following the method outlined by Dong, et al. [53]. Samples from WT, OE2, and OE16 were chosen, with each sample having three biological replicates. The calculation method adhered to protocols described previously [54].

Application of exogenous 24-epibrassinolide

Four mature plants of M. truncatula were carefully selected and sprayed with a 0.1 µM solution of EBR following the method outlined by Yuan et al. (2012) with some modifications. Samples were collected at 0 h, 0.5 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h intervals. At each time point, 0.1 g of M. truncatula leaves was swiftly frozen with liquid nitrogen and stored at -80 °C. Total RNA was extracted from the samples using the Trizol method and reverse-transcribed into cDNA for subsequent qRT-PCR analysis.

Statistical tests

The statistical data were visualized using Prism 9 software, and significance between the WT and OE samples was analyzed using the unpaired t-test.

RNA extraction and gene expression assay

Plant total RNA was extracted from 100 mg of fresh leaf, root, and shoot samples using the method described by Sun, et al. [15], with and each sample consisting of four biological replicates. Real-time quantitative RT-PCR was performed following previously published protocols [15, 55]. The ubiquitin gene of creeping bentgrass, AsUBQ (JX570760), was used as a reference. The relative expression level of each gene was calculated using the 2 −∆∆Ct method [56, 57].

RNA-seq analysis

Total RNA was extracted from each sample and treated with gDNA Eraser (TaKaRa, Japan) to eliminate contaminating genomic DNA. Samples WT and OE2 were selected, with each sample comprising three biological replicates. Details on correlations and differences among the samples are provided in the supplementary materials (Fig. S3, S4).

RNA integrity and purity were assessed on 1% agarose gels and using a NanoPhotometer spectrophotometer (IMPLEN, USA). RNA concentration was measured with a Qubit RNA Assay Kit on a Qubit 2.0 Fluorometer (Life Technologies, USA), and RNA integrity was evaluated using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 system (Agilent Technologies, USA). For library construction, 3 µg of RNA per sample was used. Sequencing libraries were generated using the VAHTSTM Universal DNA Library Prep Kit for Illumina® V2 (NuoWeiZan, China), following the manufacturer’s instructions, with index codes added to attribute sequences to each sample. Library quality was assessed using the Agilent Bioanalyzer 2100 system, and sequencing was conducted on a HiSeq4000 instrument.

After removing reads containing adapter sequences and filtering out low-quality reads from raw data, clean data were obtained and used for transcriptome analysis. RSEM was employed to estimate gene and isoform expression levels using the clean data, with the unigene sequence file as the reference gene file [58, 59]. DESeq2 software was utilized to analyze differentially expressed genes, using parameters of adjusted p-value < 0.01 and |log2 FC| >= 2 [60].

Unigene sequences were aligned to various databases, including Nonredundant protein (NR), SwissProt, Pfam, Gene Ontology (GO), Cluster of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups Database (eggNOG), and Cluster of Orthologous Groups (KOG/COG) databases [61, 62]. GO annotation was performed using Blast2GO software (version 2.3.5). The KEGG Automatic Annotation Server (KAAS1) was used for KEGG pathway annotation, and the Pfam database was searched using HMMER software (Eddy 1998).