The Influence of Bud Positions on the Changes in Carbohydrates and Nitrogen in Response to Hydrogen Cyanamide During Budbreak in Low-Chill Kiwifruit

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript investigates how the application of hydrogen cyanamide (HC) affects dormancy breaking, floral bud production, and shoot development in low-chill kiwifruit cultivated under tropical high-altitude conditions with insufficient chilling — among other aspects related to phenological responses and changes in carbohydrate and nitrogen concentrations in bark tissues at different bud positions along the branch.  I provide the following points, however, in order to increase the improvement aspect:

Citing a significant and higher number of recent articles is important;

In the abstract, the part stating that "HC induced an earlier decline in soluble sugar concentrations from the apical to basal positions" may create ambiguity. It suggests that HC induced a decline that varies from the apical to the basal position, or that it accelerated the decline by following an apical-basal gradient. The results indicate that hexose concentrations (fructose and glucose) were significantly lower in the apical position under both treatments and were generally lower in HC-treated branches across all positions. More pronounced decline in glucose at the apical position of HC-treated branches was also mentioned at 7 DAT. The abstract could be more precise in describing this temporal and spatial dynamic, perhaps emphasizing that HC accelerates the decline (utilization) especially in the apical position, where the response is eventually strongest. Maybe including in the abstract the percentage increase in dormancy breaking at the apical (58.82%) in addition to the middle (375%) would also make HC’s impact more evident across all responsive positions.

The introduction could perhaps elaborate a bit more on the specific consequences of heterogeneity and low productivity for the commercial cultivation of kiwifruit in these regions, reinforcing the practical relevance of the study. The mention that soluble sugars are involved in the regulation of apical dominance is an important point that connects with the discussion on budbreak uniformity; this connection could be briefly anticipated as one of the potential mechanisms to be explored.

The methodology for sampling could be more specific and in a explanatory way regarding the number of branches or buds sampled per position and time point for biochemical analyses (carbohydrates and nitrogen), in addition to clearly stating whether “four replications” or “three replications” were used in the results. The text does not specify how many branch pairs were used in total or per treatment/sample for the biochemical analyses over time. For phenological observations, and indeed indicating the number of buds evaluated per branch or per position would also be helpful.

I also found that the manuscript lacks more actual figures showing real stages and aspects of the experiments throughout the manuscript, especially in the methodology.

An important discussion point is the link between the lower concentrations of soluble sugars (hexoses) in the apical position and in HC-treated branches and the idea of “accelerated utilization,” which, although plausible, is an inference based on the observed correlation. The discussion could explicitly acknowledge that concentration measurement is a snapshot, and that actual transport or consumption rates were not measured in depth. Briefly exploring other possible reasons for lower concentrations (e.g., transport rates away from the sampled tissue) or more strongly justifying why “accelerated utilization” is the most likely interpretation—perhaps referencing more studies on carbohydrate usage dynamics during budbreak—could further deepen the mechanistic analysis.

Author Response

Comment1: Citing a significant and higher number of recent articles is important

Response:

Thank you for this suggestion. We have carefully reviewed the manuscript and updated the reference list by including several recent and relevant studies published to strengthen the scientific context and ensure that the discussion reflects current research in the field. These newly added references have been cited in the Introduction and Discussion sections where appropriate. Specifically, we have included the following recent studies in the revised manuscript:

[22] Bertheloot et al., 2019 – on the roles of sugars and plant hormones in regulating apical dominance

[29] Li et al., 2020 – on nitrogen storage and its dynamics during budbreak in woody plants

[42] Cao et al., 2023 – on the interaction between sugar availability and hormonal signaling in bud outgrowth

[44] Liang et al., 2019 – on the effect of hydrogen cyanamide in promoting sugar-metabolizing enzyme activity in buds

[47] Hubmann et al., 2023 – on the enhancement of sink strength in buds during budbreak

Comment2: In the abstract, the part stating that "HC induced an earlier decline in soluble sugar concentrations from the apical to basal positions" may create ambiguity. It suggests that HC induced a decline that varies from the apical to the basal position, or that it accelerated the decline by following an apical-basal gradient. The results indicate that hexose concentrations (fructose and glucose) were significantly lower in the apical position under both treatments and were generally lower in HC-treated branches across all positions. More pronounced decline in glucose at the apical position of HC-treated branches was also mentioned at 7 DAT. The abstract could be more precise in describing this temporal and spatial dynamic, perhaps emphasizing that HC accelerates the decline (utilization) especially in the apical position, where the response is eventually strongest. Maybe including in the abstract, the percentage increase in dormancy breaking at the apical (58.82%) in addition to the middle (375%) would also make HC’s impact more evident across all responsive positions.

Response:

We thank the reviewer for this insightful and constructive comment. We agree that the original wording in the abstract could lead to ambiguity regarding the spatial and temporal pattern of sugar decline following HC application. To address this, we have revised the sentence to clearly reflect that the decline in soluble sugars—particularly sucrose and hexose—was more pronounced at the apical position in HC-treated canes and that this pattern may reflect enhanced mobilization and consumption associated with increased bud break and total flower bud. Additionally, as suggested, we have now included the percentage increase in budbreak at the apical position (58.82%) to better highlight HC's differential effect across bud positions.

Revisions highligthed in lines 21-32 of the revised manuscript:

“HC significantly increased budbreak by 58.82% at the apical position and by 375% at the middle position, with corresponding increases in total flower buds by 148.78% and 1,066.67%, respectively.  Additionally, shoot lengths were uniform among bud positions in HC-treated canes, whereas non-treated canes showed shoot length heterogeneity. Moreover, HC treatment triggered an earlier and more pronounced reduction in soluble sugars (sucrose and hexoses) concentrations along the gradient from apical to basal bud positions, where the response is strongest at the apical position, which was strongly associated with enhanced budbreak percentages and total flower bud formation. While total nitrogen content was highest in the apical position, it was unaffected by HC application. These findings indicate that HC may promote budbreak by enhancing mobilization and consumption of soluble sugars for bud growth, thereby improving budbreak performance, flower bud production, and uniform shoot development in low-chill kiwifruit under warm conditions.”

Comment3: The introduction could perhaps elaborate a bit more on the specific consequences of heterogeneity and low productivity for the commercial cultivation of kiwifruit in these regions, reinforcing the practical relevance of the study.

Response:

Thank you for this valuable suggestion. In response, we have revised the Introduction to more clearly articulate the commercial implications of budbreak heterogeneity and low productivity in low-chill kiwifruit cultivation. Specifically, we added the following text (lines 45–54): “In response, low-chill kiwifruit cultivars have been introduced as an alternative, particularly in regions where insufficient winter chilling is anticipated. ‘Bruno’ is recognized as a low-chill kiwifruit cultivar but still requires approximately 750 chilling hours to adequately fulfill its dormancy release requirement and around 950 chilling hours to initiate floral emergence [6]. In the highland tropical climate of northern Thailand, where average chilling hours are approximately 350 [7], suboptimal chilling conditions severely constrain budbreak, flower bud development, and ultimately yield. These limitations are reflected in the low average budbreak per cane, reaching only 13.9% [8], compared to up to 59% under optimal chilling conditions [9].”

 

 

Comment4: The mention that soluble sugars are involved in the regulation of apical dominance is an important point that connects with the discussion on budbreak uniformity; this connection could be briefly anticipated as one of the potential mechanisms to be explored.

Response:

Thank you for this insightful suggestion. We agree that the involvement of soluble sugars in regulating apical dominance is an important point that connects well with our discussion on budbreak uniformity. In the original discussion, the interaction between plant growth-regulating hormones and sugar availability was not sufficiently addressed. To strengthen this aspect, we have added the following paragraph to the revised manuscript (lines 399-419):

 “Apical dominance has traditionally been attributed to the action of auxin, which promotes vigorous growth of the terminal bud while suppressing the outgrowth of axillary buds located at lower nodes [41]. However, recent studies have expanded this understanding by emphasizing the role of sugars in modulating auxin-related apical dominance. Specifically, the strong sugar demand of the apical bud restricted sugar translocation to axillary buds, thereby inhibiting their outgrowth due to limited sugar availability [20]. When overall sugar status in the plant was high, increased sugar availability weakened apical dominance and promoted axillary bud outgrowth [22]. Elevated sugar availability, in combination with cytokinins, has been shown to significantly counteract auxin-mediated inhibition of bud outgrowth by suppressing strigolactone signaling. Bud outgrowth is quantitatively regulated by the interplay among sugar availability, cytokinin activity, and auxin signaling trough strigolactone. [22, 42]. Further insight into the effects of specific soluble sugars revealed that sucrose, glucose, and fructose all triggered bud outgrowth and antagonized the effects of auxin and strigolactone, with glucose and fructose being more effective than sucrose [22].

Previous studies have reported that hydrogen cyanamide (HC) increases sugar availability by enhancing the activity of enzymes involved in sugar metabolism, thereby promoting the conversion of starch into soluble sugars [16, 43, 44]. Building upon this understanding, HC application may facilitate axillary budbreak and attenuate apical dominance in low-chill kiwifruit by stimulating sugar metabolic processes and increasing availability of soluble sugars.”

 

 

Comment5: The methodology for sampling could be more specific and in an explanatory way regarding the number of branches or buds sampled per position and time point for biochemical analyses (carbohydrates and nitrogen), in addition to clearly stating whether “four replications” or “three replications” were used in the results. The text does not specify how many branch pairs were used in total or per treatment/sample for the biochemical analyses over time.

Response:

To clarify the sampling procedure, we have revised the Materials and Methods section to provide a more detailed description of the number of branches sampled per time point and how samples were pooled and assigned to biochemical analyses. The following text has been added in lines 173-180:

“For biochemical analyses, three pairs of canes (three HC-treated and three non-treated) were collected from each vine and pooled to form one replicate. At each time point, six replicates were obtained, corresponding to the six vines used for phenological observations. Four replicates were used for the analysis of non-structural carbohydrates, while three were analyzed for nitrogen content. The remaining samples were retained as spare. The protective bark tissues surrounding the buds, along with the internodal bark adjacent to each bud, were collected. All bark tissues were immediately frozen in liquid nitrogen and stored at –20 °C until further analysis.”

Comment6: For phenological observations, and indeed indicating the number of buds evaluated per branch or per position would also be helpful.

Response:

Thank you for this helpful suggestion. To address this, we have revised the Methods section to clearly specify the number of canes and buds evaluated for phenological observations. The following text has been added in line 149-152:

“Five pairs of canes, comprising five HC-treated and five non-treated canes, were randomly selected from each of the six vines. Budbreak, reproductive budbreak, shoot type, total flower bud, and shoot length were recorded separately for each cane. Data were then averaged across all 30 canes per treatment, regardless of the vine origin”

Additionally, to clarify how bud positions were designated, we added the following explanation in lines 127-132:

“To ensure clear differentiation among bud positions along the cane, specific buds were consistently selected to represent each position. The first, third, and fifth buds from the distal end were designated as the apical, middle, and basal positions, respectively. This approach enabled consistent spatial separation and facilitated accurate comparisons of phenological and biochemical responses.” These additions provide a more detailed and transparent description of the phenological data collection process.

Comment7: I also found that the manuscript lacks more actual figures showing real stages and aspects of the experiments throughout the manuscript, especially in the methodology in page.

Response:

Thank you for this valuable suggestion. To improve the clarity and visual presentation of the experimental setup and bud developmental stages discussed in the manuscript, we have added two new figures:

• Figure 1 provides a schematic diagram outlining the overall experimental methodology, including cane selection, treatment application, sampling procedure, and the phenological observation parameters measured.
• Figure 2B presents actual images of bud developmental stages as referenced in the text, helping to illustrate the phenological stages assessed during the study.

 

Comment8: An important discussion point is the link between the lower concentrations of soluble sugars (hexoses) in the apical position and in HC-treated branches and the idea of “accelerated utilization,” which, although plausible, is an inference based on the observed correlation. The discussion could explicitly acknowledge that concentration measurement is a snapshot, and that actual transport or consumption rates were not measured in depth. Briefly exploring other possible reasons for lower concentrations (e.g., transport rates away from the sampled tissue) or more strongly justifying why “accelerated utilization” is the most likely interpretation—perhaps referencing more studies on carbohydrate usage dynamics during budbreak—could further deepen the mechanistic analysis.

Response:

We appreciate the reviewer’s thoughtful and constructive comment. We acknowledge that the interpretation of “accelerated utilization” is based on correlative evidence and that soluble sugar concentrations represent only a temporal snapshot rather than direct measurements of sugar transport or metabolic consumption. In response, we have revised the discussion to enhance clarity regarding the term “accelerated utilization,” which may have previously been ambiguous. While the core interpretation remains unchanged, we have expanded the explanation to clarify that “utilization” encompasses both the mobilization of soluble sugars from adjacent bark tissues toward developing buds and their subsequent metabolic consumption within those buds. The revised paragraphs are provided in the Discussion section (Lines 446–491).

“Although measurements of soluble sugars (sucrose and hexoses) concentration provided a temporal snapshots of carbohydrates status, the actual rates of transportation or metabolic consumption was not deeply assessed. Consequently, the significantly lower soluble sugar levels observed under conditions of either the apical position or HC application indicates merely that less sugars remained in the sampled tissues at the time of analysis. We attributed this to enhanced mobilization and consumption of soluble sugars for bud growth.

The enhanced mobilization of soluble sugars may involve with enzymatic processes associated with sink activity. Cell-wall invertase plays a critical role in carbohydrate partitioning and sink-strength establishment by hydrolyzing sucrose into glucose and fructose. During budbreak, elevated activity of cell-wall invertase strengthens sink capacity in developing buds [16, 43, 45, 47], facilitating the uptake of soluble sugars from surrounding tissues and consequently reducing local sugar concentrations. Consistent with this explanation, soluble sugar concentrations in internode tissues were significantly reduced following HC application in grapevine, coinciding with increased cell-wall invertase activity in the buds [43]. Furthermore, previous studies reported that sugar influx into bark tissues declined significantly [45] or remained substantially lower than that into developing buds during budbreak, with apical buds exhibiting markedly higher influx rates compared to basal buds [17]. This highlights the stronger sink activity of apical buds during the budbreak period.

The increased metabolic activation and sugar consumption involves the early and pronounced decrease in sucrose concentration, accompanied by a concurrent rise in hexose levels following HC application, indicating a key sign of metabolic reactivation associated with the resumption of bud growth in kiwifruit [31]. HC treatment enhanced sugar metabolism at the transcriptional level by upregulating key enzymes such as sucrose synthase, hexokinase, and amylases, which promoted early starch degradation and increased soluble sugar availability [44]. As a result, soluble sugar concentrations declined significantly and showed an earlier depletion pattern in HC-treated buds [43-44]. These findings suggest that HC application stimulated metabolic activity and enhanced early soluble sugar consumption for supporting bud growth.

 The alternative explanation could be that carbohydrate reserves were initially low either at the apical position or before HC treatment seems unlikely. Specifically, soluble sugar measurements taken 25 days before HC application revealed that sucrose concentrations were highest at the apical position (Figure S1), whereas hexose concentrations were relatively similar across all bud positions (Figure S2). These observations suggest that the significant differences in soluble sugar concentrations likely progressed following HC application or bud development rather than reflecting initial lower soluble sugar concentrations.

Taken together, this study demonstrated that significantly lower soluble sugar concentrations in bark tissues, observed under both apical positioning and HC application, were correlated strongly with increased budbreak percentage and total flower buds. These findings suggest that HC treatment may promote rapid mobilization of soluble sugars toward developing buds, along with their early subsequent metabolic consumption. This sink-driven mobilization of carbohydrates may contribute to the depletion of soluble sugars in adjacent tissues such as bark, supporting energy supply and metabolic reactivation necessary for bud development and the initiation of budbreak.”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper presents a solid and well-structured study on the effects of cyanamide (HC) application on sprouting of kiwifruit with low chilling requirements at tropical altitudes. The proposal is relevant and original as it links the changes in carbohydrates and nitrogen in the bark tissues to the position of the buds and phenological development. The introduction adequately contextualizes the problem of insufficient cold accumulation in a climate change scenario and justifies the need for the study. However, it is recommended to provide a more explicit working hypothesis at the end of this section to facilitate the understanding of the experimental objective.
The methodology used is appropriate and well described so that replicability is possible. The choice of experimental site and cultivar ‘Bruno’ is well justified, although it would be appropriate to explain why samples were only taken up to 28 days after treatment when phenological observations were made up to the 55th day. The results are well presented and statistically validated. There is a clear correlation between the decrease in soluble sugars and the increase in sprouting, especially in the apical and middle positions. However, the interpretation of the fluctuations in nitrogen content requires further clarification.

The discussion is well organized and supported by current and relevant literature. The patterns of carbohydrate accumulation and consumption are properly emphasized, as is their relationship to germination.

- However, it would be helpful if the authors indicated whether the enzymatic activities related to hydrolysis of starch and sugars have been measured, as they are mentioned in the discussion but no experimental data are presented.

• 
  - It would be interesting to suggest complementary studies on the interaction between carbohydrates and growth-regulating hormones in this context.
• The article is well written, with an appropriate use of technical English and a clear style. There are only a few passages that could benefit from minor editing to improve the flow of the text.

Author Response

Comment1: However, it is recommended to provide a more explicit working hypothesis at the end of this section to facilitate the understanding of the experimental objective.

Response:

To provide a more explicit working hypothesis, we have added text to the revised manuscript in lines 100-103 “This study is based on the hypothesis that the protective bark tissue adjacent to developing buds may undergo temporal changes in non-structural carbohydrates and total nitrogen contents during the budbreak period. These patterns may be influenced by hydrogen cyanamide (HC) application and bud position.”

 

Comment2: The choice of experimental site and cultivar ‘Bruno’ is well justified, although it would be appropriate to explain why samples were only taken up to 28 days after treatment when phenological observations were made up to the 55th day.

Response:

To clarify this point, we have indicated that sampling for biochemical analysis was conducted up to 28 days after treatment (DAT), as this time point corresponded to when budbreak at the apical position of HC-treated canes reached approximately 50% (as mentioned in line 239-240). This stage was considered the most appropriate for detecting differences in carbohydrate and nitrogen concentrations between treatments and bud positions, as bud developmental stages were clearly differentiated at this time.  

In contrast, phenological observations were extended to 55 DAT to capture the stage at which budbreak at the apical position of non-treated canes also reached 50%. As mentioned in the manuscript, “At 55 DAT, approximately 50% of the apical buds under the non-treated condition had been released from dormancy. At this stage, shoot development had progressed sufficiently to allow for the assessment of budbreak incidence, flower bud counts, and shoot length. Distinct differences in phenological development between HC-treated and non-treated canes were apparent” in lines 134-138. We also provide a visual representation of shoot development during this period in Figure 2A. 

Comment3: However, the interpretation of the fluctuations in nitrogen content requires further clarification.

Response:

To expand the clarification, we have now added the following paragraph to the revised manuscript in lines 492-506:

“In HC-treated canes, the decline in total nitrogen content in the bark tissues at the apical position during the early sampling period was likely attributable to the remobilization of stored nitrogen to support the development of emerging shoots [29]. Subsequently, total nitrogen content slightly increased during the budbreak period, aligning with trends previously reported in kiwifruit [15]. Among all bud positions, the apical position consistently exhibited the highest nitrogen levels in both HC-treated and non-treated canes. According to [29] reduced accumulation of bark storage proteins (BSPs) was associated with limited early growth and delayed budbreak in poplar trees. Furthermore, elevated nitrogen concentrations, particularly in the form of ammonium and protein, have been associated with enhanced floral development in Prunus species [48]. In line with earlier studies, current finding showed that higher total nitrogen contents at apical position was associated with significantly higher budbreak percentage and flower bud production. Moreover, total nitrogen contents showed minimal differences between HC and non-treated canes across all bud positions, indicating that nitrogen content was not notably affected by HC application.”

Comment4: However, it would be helpful if the authors indicated whether the enzymatic activities related to hydrolysis of starch and sugars have been measured, as they are mentioned in the discussion, but no experimental data are presented.

Response:

We agree with the reviewer that enzymatic activities related to starch and sugar hydrolysis are important indicators of metabolic processes in bark tissues. However, the exact same samples used for biochemical analysis during the 2007–2008 growing season were completely used up in those assays. Additionally, enzymatic activity measurements require a different sample preparation protocol. As a result, it was not possible to conduct enzyme assays from the same set of samples. Nonetheless, recognizing the importance of this information, we have incorporated enzymatic activity measurements into a subsequent experiment using newly collected samples from both bud and bark tissues to further investigate the underlying physiological processes.

 

 

Comment5: It would be interesting to suggest complementary studies on the interaction between carbohydrates and growth-regulating hormones in this context.

Response:

Thank you for this insightful suggestion. We acknowledge that the interaction between plant growth-regulating hormones and sugar concentration, particularly in relation to apical dominance, was not sufficiently addressed in the original discussion. To strengthen this aspect, we have now added the following paragraph to the revised manuscript in lines 399-419:

“Apical dominance has traditionally been attributed to the action of auxin, which promotes vigorous growth of the terminal bud while suppressing the outgrowth of axillary buds located at lower nodes [41]. However, recent studies have expanded this understanding by emphasizing the role of sugars in modulating auxin-related apical dominance. Specifically, the strong sugar demand of the apical bud restricted sugar translocation to axillary buds, thereby inhibiting their outgrowth due to limited sugar availability [20]. When overall sugar status in the plant was high, increased sugar availability weakened apical dominance and promoted axillary bud outgrowth [22]. Elevated sugar availability, in combination with cytokinins, has been shown to significantly counteract auxin-mediated inhibition of bud outgrowth by suppressing strigolactone signaling. Bud outgrowth is quantitatively regulated by the interplay among sugar availability, cytokinin activity, and auxin signaling trough strigolactone. [22, 42]. Further insight into the effects of specific soluble sugars revealed that sucrose, glucose, and fructose all triggered bud outgrowth and antagonized the effects of auxin and strigolactone, with glucose and fructose being more effective than sucrose [22].

Previous studies have reported that hydrogen cyanamide (HC) increases sugar availability by enhancing the activity of enzymes involved in sugar metabolism, thereby promoting the conversion of starch into soluble sugars [16, 43, 44]. Building upon this understanding, HC application may facilitate axillary budbreak and attenuate apical dominance in low-chill kiwifruit by stimulating sugar metabolic processes and increasing availability of soluble sugars.”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been improved. I have no more suggestions at this time