Trimming induces tillering in bermudagrass
First, the tillering process in the main stem and stolon node was measured 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 was extended. On the 10th day of treatment, the control group had 9 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 (Fig.1C and D). The pattern of dynamic change of the 2nd buds in response to trimming was estimated. 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 (Fig.1E and F). Throughout the treatment, there was no significant change in the length of tiller buds in intact stolons, while the length of tiller buds showed a S-shaped growth pattern after trimming. These results showed 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 hours and reached its lowest expression level at 12 hours, indicating a consistent effect of trimming on stolon tiller bud induction. (Fig.2A). In terms of spatial expression, TB1 was significantly down-regulated at the 2nd and 3rd nodes after 6 hours of trimming, compared to the control (Fig.2B).
Trimming induced temporal and spatial changes in CTK biosynthesis
Given the fact that CTK is induced by decapitation and promotes bud outgrowth, the changes in CTK biosynthesis in response to trimming were estimated. The active form of CTK and zeatin (ZT) was found to have accumulated significantly only at the 1st node of the stolon (Fig. 3A). The spatial expression pattern of the genes related to CTK biosynthesis showed that LONELY GUY1(LOG1) and ISOPENTYL TRANSFERASE1(IPT1) were significantly up-regulated at the 1st and 2nd nodes 6 hours after trimming treatment (Fig.3B and C). Furthermore, the time-course expression of these genes at the 1st node showed that LOG1 was rapidly up-regulated by 3.1 times 1 hour after trimming treatment and maintained this expression level until 12 hours after trimming (Fig.3D). IPT1 was slightly induced at the early stage of treatment and peaked at 12 hours after treatment, showing a 273-fold increase (Fig.3E). Therefore, it appears that trimming stimulated the accumulation of CTK, which was dependent on the node position.
6-BA was exogenously sprayed onto the stolons, resulting in the induction of tiller buds at the 3rd, 4th, and 5th nodes (Supplementary Fig1).
Trimming increased the energy distribution towards the tillering process by enhancing photosynthesis
Tillering is an energy-consuming process. To determine whether photosynthesis was involved in the response to trimming, the photosynthesis-related parameters were measured.
The OJIP fluorescence transient curve exhibited an elevation at both 6 and 24 hours after trimming, with a greater enhancement observed at 24 hours compared to 6 hours (Fig. 4A). To further study the effect of trimming on the photosynthetic system, we derived additional parameters through the JIP test (Supplementary Tab.1). It is evident that some parameters were enhanced after treatment. Notably, basic photosynthetic parameter(M0), quantum yield parameter (φEo) and efficiency parameter (Ψ0), as well as performance index PIcs (p<0.01) showed highly significant differences after 6 hours of trimming. The performance indices PIABC, PITotal, and PIcs can more accurately reflect the state of the photosynthetic mechanism, and as seen in Supplementary table1, trimming can increase these indices.
Sucrose is the end product of photosynthesis, while 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 estimating 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 (Fig. 5A and B), and fructose content was remarkably higher at the 3rd node compared to the control (Fig.5C). Moreover, SPS was notably up-regulated at the 1st, 2nd, and 3rd nodes of the stolon compared to the control 6 hours after trimming treatment (Fig.5D). The time-course expression analysis of SPS at the 3rd node revealed that it was up-regulated from 3 hours after trimming and reached its peak at 12 hours, exhibiting a 3.7-fold increase compared to the control group (Fig.5E). Furthermore, the exogenous spraying of sucrose induced the formation of tiller buds (Supplementary Fig1).
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 hour of trimming and remained elevated for 24 hours (Fig.6A). Moreover, the application of exogenous H2O2 resulted in a 1.7-fold increase in the number of tillers in intact plants (Fig.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 application of exogenous 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 consider 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 24-72 hours of trimming treatment. Importantly, exogenous H2O2 further enhanced the antioxidant enzymes activity triggered by trimming (Fig. 8A, B and C).