H₂O₂ promotes trimming-induced tillering by regulating energy supply and redox status in bermudagrass

Plant material and growth conditions

The experimental material used was bermudagrass ‘A12359’ (2n = 36), which was planted in 30 pots (diameter: 15.5 cm, height: 17.5 cm) on November 3, 2021, and cultivated in the greenhouse of Binhai Grass Germplasm Resources Breeding Base located in the northern area of Ludong University, Yantai City, Shandong Province (121°36′N, 37°53′E). It received watering every two days and was provided with fresh 1/2-strength Hoagland’s nutrient solution (formula: NH₄H₂PO₄ (0.5 mM), KNO₃ (2.5 mM), Ca (NO₃)₂·4H₂O (2.5 mM), MgSO₄·7H₂O (1 mM), H₃BO₃ (23 µM), ZnSO₄·7H₂O (0.38 µM), CuSO₄·5H₂O (0.16 µM), MnCl₂·4H₂O (4.5 µM), H₂MoO₄ (0.2 µM), Fe-EDTA (25 µM).) once a week to ensure adequate supply of water and nutrients. After the bermudagrass had developed abundant stolons, it was decapitated.

Trimming treatment

The experimental materials were cultivated to have two tillers and then trimmed. During each treatment, bermudagrass was uniformly trimmed using auxiliary tools to maintain a stubble height of 5 cm. Both control and treatment groups consisted of six biological replicates. Tiller buds containing two leaves and one central bud were defined as tillers. From the beginning of the experiment until 10 days after treatment, the number of tillers was counted every 2 h. At 2, 4, 6, 8 and 10 h after treatment, the number of tillers on the main stem of bermudagrass in both the control and treatment groups were counted multiple times, and the dynamic change curve of the number of tillers in response to grass trimming was plotted. When bermudagrass produces more stolons, the stolons with uniform growth were selected and labeled accordingly. Two groups, control and trimming, each consisting of five stolons, were established. Trimming was simulated by removing the topmost stem node. From the beginning of the experiment to 6 days after treatment, the length of tiller buds at different nodes was measured daily. Subsequently, tiller buds at the second node of the control and trimming groups were compared to analyze the pattern of their dynamic changes in response to trimming.

Exogenous treatment

Three pots of bermudagrass exhibiting similar growth were divided into three groups: one group was sprayed with 50 µM 6-Benzylaminopurine (6-BA), the second group was sprayed with 200 mM sucrose, and the third group was sprayed with distilled water as a control. To measure the length of tiller buds at the end of the treatment, six stolons were selected for each treatment. The treatments were performed every other day for a total of six times.

Four treatment levels were established for bermudagrass, including a non-trimming group consisting of a control and exogenous H₂O₂, and a trimming group consisting of a control and exogenous H₂O₂, with six replicates per treatment level. Bermudagrass with a consistent initial tiller count of four was selected. The leaves were treated with 2 mM H₂O₂ until they were dripping, and this process was repeated every other day (concentrations were determined by pre-experimentation). The tillers were counted after each treatment until the 8th day.

Gene expression analysis

From each of the first four nodes of bermudagrass stolons, 0.1 g was taken after 6 h of trimming treatment. 0.1 g of nodal sample was taken after 0, 1, 3, 6, 12, and 24 h upon trimming treatment. Additionally, 0.1 g of nodal sample was taken after exogenous H₂O₂, including four treatment levels. Three replicates of each sample were ground to powder in liquid nitrogen. Total RNA was extracted using the Plant RNA Kit with genomic DNA enzyme (Vazyme Biotech Co., Ltd, Jiangsu, China), and the RNA concentration and quality were measured using a NanoDrop microspectrophotometer (Eppendorf, Barkhausenweg, Germany). Subsequently, 5 µL of RNA was reverse transcribed to obtain cDNA with HiScript II 1st Strand cDNA Synthesis Kit with genomic DNA enzyme (Vazyme Biotech Co., Ltd, Jiangsu, China), which was then employed as a template for qRT-PCR (Quantitative real-time reverse transcription-PCR, Thermo Fisher Scientific, Waltham, MA, USA) to detect gene expression using SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Jiangsu, China).

Zeatin and sugar measurement

After 24 h of trimming, nodes were collected and freeze-dried in liquid nitrogen, then stored at −80 °C until needed. The dried plant materials were then homogenized and powdered in a mill.

For zeatin detection, 100 mg of dried powder was extracted with 1.5 mL of a mixed solution of MeOH: H₂O: FA (79.9: 20: 0.1). The extract was vortexed and subjected to ultrasound for 30 min, then stored at 4 °C for 12 h. After centrifugation, supernatants were collected. The residue was reextracted with one mL of MeOH under ultrasound for 30 min and centrifuged. The supernatants were combined, evaporated to dryness under a nitrogen gas stream, and reconstituted in 100 µL of a mixed solution of MeOH: H₂O (50: 50).

For sugar detection, 30 mg of dried powder was extracted with a mixed solution of Ethanol: H₂O (80: 20) in a volume of 700 µL. The extract was vortexed and subjected to ultrasound for 30 min, placed in a 70 °C water bath, and kept there for 2 h. Next, 700 µL of chloroform was added and supernatants were collected after centrifugation. The supernatants were then evaporated to dryness under a nitrogen gas stream and reconstituted in a mixed solution of ACN: H₂O (75: 25) in a volume of 100 µL.

Finally, the solution was filtered through a 0.22 µm filter for further LC-MS analysis.

Phytohormones contents were detected by Guocangjian (http://www.targetcrop.com/) based on the Sciex 4500 LC-MS/MS platform.

OJIP fluorescence transient test

After trimming the first node of bermudagrass stolon for 6 and 24 h, intact leaves (the two foremost leaves of the stolon) and the leaves of newly formed tillers were collected in each treatment, which was repeated six times. The samples to be tested were subjected to the dark treatment for 30 min before being measured by a chlorophyll fluorimeter (PAM2500). The leaves of H₂O₂ exogenous treatment at the four treatment levels were also measured using a chlorophyll fluorometer (PAM2500) to obtain OJIP fluorescence transient curves.

Starch content determination

After trimming the stem nodes for 0 and 24 h, the leaves of newly formed tillers were sampled, dried, and weighed (0.5 g). Each treatment was replicated three times. A total of 6–7 ml of 80% ethanol was added to the sample, followed by extraction in a water bath maintained at 80 °C for 30 min before centrifugation. This extraction process was repeated twice, and the precipitate was collected. Next, 3 ml of distilled water was added to the precipitate, which was then boiled in a water bath for 15 min. After cooling, 2 ml of chilled 9.2 M perchloric acid was added, and the sample was extracted for 15 min and centrifuged. The precipitate was then mixed with 4.6 M perchloric acid for 15 min and centrifuged. The precipitate was washed twice, and combined with centrifugal liquid to make a constant volume of 50 ml. A total of 1 ml of the extract and 5 ml of anthrone were mixed and shaken well in a boiling water for 10 min. The mixture was then cooled and the wavelength was measured at 625 nm with a spectrophotometer to calculate the starch content (Ahamed et al., 1996).

H₂O₂ content determination

After being subjected to trimming treatment for 0, 1, 3, 6, 12, 24, 48 and 72 h, the sample was weighed at 0.1 g. Subsequently, nine times the volume of PBS (pH 7.0–7.4, 0.1 mol/L) was added following a weight (g) to volume (mL) ratio of 1:9. The supernatant homogenate was mechanically shaken under an ice-water bath and collected for determination using a hydrogen peroxide (H₂O₂) test kit (Jiancheng, Nanjing, China).

Antioxidant enzyme activity assay

After treatment, 0.25 g of the sample was ground in liquid nitrogen. Subsequently, 4 ml of phosphate buffer (150 mM, pH =7.0) was added, and the mixture was centrifuged at 12,000 r/min and 4 °C for 20 min. The resulting supernatant was considered as the crude enzyme extract.

The activity of superoxide dismutase (SOD) was measured according to the method previously described, which was based on the reduction of its inhibition to nitro blue tetrazolium (NBT) (Dhindsa, Plumb-Dhindsa & Thorpe, 1981). For peroxidase (POD) activity assay, we followed the procedure outlined by Pütter & Becker (1983). Catalase (CAT) activity was determined essentially according to the method described by EI-Moshaty (El-Moshaty et al., 1993). The decrease in H₂O₂ was monitored at 240 nm and quantified using the molar extinction coefficient of 36 M⁻¹ cm⁻¹.

Trimming induces tillering in bermudagrass

To determine the effect of trimming on tillering in bermudagrass, we first measured the tillering process in the main stem and stolon node in response to trimming. Two days after trimming, a significant difference in tiller count was observed in the main stem compared to the control group, and this difference became more pronounced as the treatment period extended. On the 10th day of treatment, the control group had nine tillers while the trimming group had 13 tillers, representing a 44% increase in tiller generation resulting from trimming (Fig. 1B). In addition, trimming stimulated the outgrowth of tiller buds located at the morphologically upper 1st, 2nd, and 3rd nodes of the stolon, with the most significant effect observed at the 1st node. However, trimming could not promote the growth of tiller buds at the 4th node (Figs. 1C and 1D).

We further analyzed the dynamic change pattern of the 2nd buds in response to trimming. It was found that on the second day after treatment, tiller buds appeared longer compared to the control, and the difference gradually increased with the extension of treatment time. On the 6th day of treatment, the length of the tiller buds that underwent trimming treatment was 3.2 times longer than the control (Figs. 1E and 1F). Throughout the treatment, there was no significant change in the length of tiller buds in intact stolons, while the length of tiller buds showed an S-shaped growth pattern after trimming. These results indicated that the sprouting and growth of tiller buds occurred earlier in the trimming group than in the control group.

After trimming treatment, the marker gene TEOSINTE BRANCHED1 (TB1), which inhibits bud outgrowth, was down-regulated after 6 h and reached its lowest expression level at 12 h, indicating a consistent effect of trimming on stolon tiller bud induction (Fig. 2A). Regarding organ-specific expression, TB1 was significantly down-regulated at the 2nd and 3rd nodes after 6 h of trimming compared to the control (Fig. 2B).

Trimming induced temporal and spatial changes in CKs biosynthesis

Considering that CKs is induced by decapitation and promotes bud outgrowth, we assessed the changes in CKs biosynthesis in response to trimming. The active form of CKs zeatin (tZ) accumulated significantly only at the 1st node of the stolon (Fig. 3A). The spatial expression pattern of the genes related to CKs biosynthesis showed that LONELY GUY1 (LOG1) and ISOPENTYL TRANSFERASE1 (IPT1) and were significantly up-regulated at the 1st and 2nd nodes 6 h after trimming treatment (Figs. 3B and 3C). We identified 15 cytokinin oxidase/dehydrogenase (CKX) genes in the bermudagrass genome and examined their temporal response to trimming. Subsequently, we screened the two most promising genes, CKX10 and CKX12 (Fig. S1). Our results revealed a significant down-regulation of CKX10 and CKX12 at the 1st-3rd nodes following a 6 h trimming treatment (Figs. 3D and 3E). Furthermore, the time-course expression of these genes at the 1st node showed that LOG1 was rapidly up-regulated by 3.1 times 1 h after trimming treatment and maintained this expression level until 12 h after trimming (Fig. 3F). IPT1 was slightly induced at the early stage of treatment and peaked at 12 h after treatment, showing a 273-fold increase (Fig. 3G). Meanwhile, CKX10 and CKX12 exhibited a rapid down-regulation of 0.66-fold and 0.27-fold, respectively, at 1 h after trimming, followed by a sustained downward trend. (Figs. 4G and 4I). Therefore, our results indicated that trimming stimulated the accumulation of CKs, which was dependent on the node position.

Next, we investigated the effects of exogenously applied 6-BA, a synthetic cytokinin, on tiller bud induction. The results demonstrated that the exogenous application of 6-BA effectively induced tiller buds at the 2nd, 3rd, 4th, and 5th nodes (Fig. S2).

Trimming increased the energy allocation towards the tillering process by enhancing photosynthesis

Since tillering is an energy-consuming process, we sought to investigate whether photosynthesis played a role in the response to trimming by measuring various photosynthesis-related parameters. Leaves of plants show a characteristic polyphasic Chl a fluorescence induction curve under high intensity continuous light illumination, termed as the OJIPSMT transient. The OJIP fluorescence transient curve exhibited an elevation at both 6 and 24 h after trimming, with a more pronounced enhancement observed at 24 h compared to 6 h (Fig. 4A). To further study the effect of trimming on the photosynthetic system, we derived additional parameters through the JIP test (Table S2). It is evident that some parameters were enhanced after treatment. Notably, basic photosynthetic parameter (Mo), quantum yield parameter (φEo) and efficiency parameter (Ψo), as well as performance index PI_cs (P<0.01) showed highly significant differences after 6 h of trimming. The performance indices PI_ABC, PI_Total, and PI_cs, which provide a more accurate reflection of the photosynthetic state, also increased after trimming as seen in Table S2.

Sucrose is the end product of photosynthesis, and starch serves as the primary storage form of photosynthates. In line with the increased potential for photosynthesis, there was a 44.28% increase in sucrose content (Fig. 4B) and a 57.77% increase in starch content (Fig. 4C) in the leaves of newly formed tillers following trimming.

We further studied the effect of trimming on sugar levels in stolon nodes by analyzing both the sugar content and expression level of the sucrose biosynthesis gene, sucrose phosphate synthase (SPS). Our findings indicated that sucrose and glucose content significantly increased at the 3rd and 4th nodes (Figs. 5A and 5B), with fructose content notably higher at the 3rd node compared to the control (Fig. 5C). Moreover, SPS was significantly up-regulated at the 1st, 2nd, and 3rd nodes of the stolon compared to the control 6 h after trimming treatment (Fig. 5D). The time-course expression analysis of SPS at the 3rd node revealed that it was up-regulated starting from 3 h after trimming, reaching its peak at 12 h (Fig. 5E). Furthermore, exogenously spraying sucrose induced the formation of tiller buds (Fig. S2).

H₂O₂ facilitated trimming-induced tillering by enhancing photosynthesis

Given the role of H₂O₂ in controlling bud outgrowth, we examined its response to trimming and its effect on tillering. Our findings indicated that H₂O₂ levels in both the clipped leaves and newly-formed tillering leaves increased rapidly within 1 h of trimming and remained elevated for 24 h. At 48 h after treatment, H₂O₂ levels in the leaves of newborn tillers decreased to a level below that of the control and did not increase (Fig. 6A). Moreover, the exogenous application of H₂O₂ resulted in a 1.7-fold increase in the number of tillers in intact plants (Figs. 6B and 6C). Trimming stimulated tillering, which was further enhanced by the application of H₂O₂.

Chlorophyll fluorescence in the leaves of the newly-formed tillers was measured. The exogenous application of H₂O₂ led to an increase in the values represented by the OJIP curve for the leaves of untrimmed-plants, and the enhancing effects of trimming on the OJIP curve were further amplified by the application of H₂O₂ (Fig. 7A). Consistently, the application of exogenous H₂O₂ to intact plants resulted in a 1.48-fold increase in sucrose content, which was further increased by 1.55-fold upon trimming (Fig. 7B). Similarly, exogenous H₂O₂ increased starch content in intact plants, with the highest starch content observed in the trimming+H₂O₂ treatment group, showing a 1.41-fold increase compared to the trimming treatment group (Fig. 7C).

The dual role of H₂O₂ in regulating plant growth prompted us to assess the antioxidant capacity of plants treated with H₂O₂. Interestingly, the activities of SOD, POD, and CAT were enhanced by both trimming and exogenous H₂O₂ after 24 to 72 h of trimming treatment. Importantly, exogenous H₂O₂ further enhanced the antioxidant enzymes activity triggered by trimming after 48 h of exogenous treatment (Figs. 8A–8C).

Our results showed that both CKs and H₂O₂ promoted bermudagrass tillering. Therefore we considered about interplay of CKs and H₂O₂ in regulation of tillering after trimming. Exogenous H₂O₂ promotes the up-regulation of CKs biosynthetic genes IPT1 and LOG1 in intact plants. The trimming+ H₂O₂ treatment exhibited the strongest up-regulation trend, being 1.39-fold and 0.96-fold higher than the trimming treatments, respectively (Figs. 9A and 9B). Regarding CKX10 and CKX12, these genes were significantly down-regulated after exogenous H₂O₂ treatment in intact plants, with trimming+ H₂O₂ showing a 0.52-fold and 0.12-fold decrease compared to the trimming group (Figs. 9C and 9D).

Trimming triggered tillering in bermudagrass by inducing cytokinin

The development and growth of tillers have a significant impact on the overall growth of plants. Currently, decapitation has been demonstrated to induce tiller formation in many species such as pea, Arabidopsis, and rose (Morris et al., 2005; Tatematsu et al., 2005; Mason et al., 2014). However, there are fewer studies on the effect of decapitation on perennial herbs. In barley, simulated grazing through clipping revealed that a certain degree of clipping could increase the number of tillers (Yuan, Li & Yang, 2020). This finding is consistent with our results, which indicate that trimming promotes tillering in the main stem and tiller bud development in the stolon node (Fig. 1).

BRC1/TB1 is considered a key transcription factor for inhibiting lateral bud outgrowth. Multiple pathways regulating branching and tillering converge to regulate the expression of BRC1/TB1 (Takeda et al., 2003; Minakuchi et al., 2010). In a previous study conducted on bermudagrass, the transcriptional level of BRC1/TB1 was found to be negatively correlated with tiller capacity (Zhang & Liu, 2018). We observed significant inhibition of this gene at the 2nd and 3rd nodes of bermudagrass stolon after trimming treatment (Fig. 2B). These results suggest that TB1 may play a role in the trimming-induced tillering at specific node positions.

The development of tillers/branches and their formation process are regulated by interactions between multiple plant hormones. The accumulation of CKs in lateral buds and stems, as well as the expression of related genes, reflects the important role of CKs in regulating lateral bud outgrowth (Young et al., 2014). Rapid accumulation of CKs in stem nodes and axillary buds has also been observed in pea (Cao et al., 2023) and rose (Roman et al., 2016) after decapitation. Moreover, decapitation induces transient expression of CKs biosynthetic genes IPT and LOG (Cao et al., 2023; Tanaka et al., 2006). While deactivation of cytokinin is the sole responsibility of the enzyme called cytokinin oxidase/dehydrogenase, CKX (Jiang et al., 2016). Reducing CKX expression promotes the accumulation of CKs in organs, it demonstrated in rice (Ashikari et al., 2005) and barley (Zalewski et al., 2010). Some studies also shown that exogenous application of CKs oxidase/dehydrogenase inhibitors in winter wheat and spring barley increased the CKs activity (Nisler et al., 2021). In our study, both CKs content and its biosynthetic genes were found to increase only at the 1st node after trimming. The CKX gene was significantly down-regulated at 1st to 3rd node (Fig. 3). This is consistent with findings in pea, where CKs levels were significantly enhanced at the upper nodes after decapitation (Cao et al., 2023). Therefore, trimming enhances CKs accumulation in bermudagrass stolons and the accumulation of CKs induced by trimming may depend on the distance from the site of trimming.

Trimming enhanced energy supply during the tillering process

Regrowth and tillering after trimming require sufficient energy supply to the newly formed tillers (Barbier et al., 2019). We observed an improvement in the photosynthetic rate of bermudagrass after trimming, with photosynthetic products accumulating in the leaves (Fig. 4). This result suggests that trimming can promote the accumulation of energy metabolites for tiller production. Our finding is consistent with the results of studies on tea trees, where enhanced photosynthetic performance contributed to bud growth (Yue, Wang & Yang, 2021). Light and sucrose serve as signals and energy sources for bud growth, thereby regulating plant growth and development. Moreover, sugars (photosynthates) act as signaling molecules to induce axillary bud development (Signorelli et al., 2018). Decapitation is a means to rapidly induce sugar signal transduction and promote axillary bud release (Mason et al., 2014). Decapitation induces axillary bud development by reducing competition for sugars in the apical bud, leading to an accumulation of sugars in the axillary buds and thus triggering a series of pathways that induce axillary bud germination. In tobacco, the expression of sucrose biosynthesis genes, SPS and sucrose phosphate phosphatase (SPP), is up-regulated after topping (Wang et al., 2018). Similarly, after the topping of chrysanthemums, the expression levels of sucrose carriers (SUCs) and Sugars Will Eventually be Exported Transporters (SWEETs) increased (Sun et al., 2021). Meanwhile, the sucrose content increased significantly after trimming (Mason et al., 2014). Our results also showed significant up-regulation of the SPS gene and accumulation of sugar content at the 3rd and 4th nodes (Fig. 5), suggesting the potential role of sugar in trimming-induced bud development.

H₂O₂ contributed to trimming-induced tillering by enhancing photosynthesis and antioxidant capacity

ROS, particularly H₂O₂, have been demonstrated to be important regulators of bud outgrowth. Studies have shown that H₂O₂ levels remain high in the dominant bud (Chen et al., 2016; Porcher et al., 2020). In rosebush, the H₂O₂ content was found to gradually decrease only in the buds and not in the neighboring stems after decapitation, suggesting that local changes in H₂O₂ content were not a result of a systematic response to wounding (Porcher et al., 2020). On the contrary, the H₂O₂ content showed a continuous increase in both the clipped leaves and the adjacent intact leaves in the newly formed tillers (Fig. 6A), suggests that trimming-induced tillering is accompanied by early H₂O₂ accumulation. This result contradicts previous research and suggests that trimming may result in a wounding response in bermudagrass.

Exogenous application of H₂O₂ has been shown to severely reduce bud outgrowth in tomato and rosebush by increasing the H₂O₂ levels (Chen et al., 2016; Porcher et al., 2020). However, we obtained opposite results in our study, where exogenous H₂O₂ was found to promote tillering after trimming in bermudagrass (Figs. 6B and 6C). Nevertheless, the fact that bud outgrowth requires a reduced status led us to consider the effects of H₂O₂ on antioxidant capacity. Studies have shown that activation of the H₂O₂ scavenging system, such as an increase in glutathione (GSH) level, is linked to greater bud outgrowth. Furthermore, pretreatment with H₂O₂ has been shown to enhance antioxidant-related enzyme activity and improve stress resistance (Guler & Pehlivan, 2016; Sathiyaraj et al., 2014). Additionally, grazing in sheepgrass has also been shown to increase the expression of antioxidant-related genes (Huang et al., 2014). These findings indicate that early burst of H₂O₂ enhances the antioxidant enzyme activities induced by trimming, which is consistent with our results (Fig. 8). Additionally, we observed that hydrogen peroxide improved the photosynthetic properties of bermudagrass and increased the accumulation of energetic substances (Fig. 8). This aligns with previous studies that have shown exogenous H₂O₂’s ability to restore photosynthetic efficiency in tomato (Nazir, Hussain & Fariduddin, 2019) and increase sugar and starch content in melons (Ozaki et al., 2009). Moreover, H₂O₂ further upregulated the genes related to CKs biosynthesis and downregulated its degradational genes under trimming condition. These findings imply an integration between H₂O₂ and CKs in inducing tillering of bermudagrass. Taken together, it is possible that H₂O₂ acts as a second messenger, rather than an oxidized molecular, in response to trimming, helping to maintain the redox status and enhance energy supply during tiller growth. Our findings suggest that the role of H₂O₂ in controlling tillering in trimming-tolerant bermudagrass differs from its regulatory role in bud outgrowth in other species.