Effect of Phosphorylation Sites Mutations on the Subcellular Localization and Activity of AGPase Bt2 Subunit: Implications for Improved Starch Biosynthesis in Maize

Round 1

Reviewer 1 Report

Effect of phosphorylation sites mutations on the subcellular localization and activity of AGPase Bt2 subunit: Implications for improved starch biosynthesis in Maize. By Guowu et al

 

In the present manuscript, authors focus on effects of phosphorylation sites mutations on the subcellular localization and activity of AGPase Bt2 subunit.

 

I read this manuscript with interest.

 

I have only a few questions/remarks:

Please change the keywords, they are probably meant to help less savvy readers and basically only "starch synthehis" says anything, the others are incomprehensible or imprecise: "phosphorylation" of what???, subcellular localization of what?, what is Bt2 activity?

In my opinion, there are too many simplifications here, it is better to omit a few keywords and instead give precise information about what this manuscript is about

In the "Introduction", the authors describing the synthesis of starch should mention the role of starch phosphorylase (SP) [39], especially since they mention this enzyme in the discussion.

 

It is worth mentioning that the publication is written very carefully and without typos, so in the section "Materials and methods" in chemical formulas numbers should be formatted with appropriate upper or lower indices, e.g., in Na₂CO₃ and other chemical formulas: 2.4, 2.6, 2.7, 2.8

Line 68:  instead of "ADPG transporter" should be ADP-Glc transporter

Line 375: instead of [40] should be [39]

Overall, I find the paper very interesting and relevant for a better understanding of AGPase activation. If there were other aspects that I think would improve the significance of the work, they would be bioinformatics analyzes and modeling of AGPase subunit structures as a function of sequence and possibly phosphorylation of the phosphorylation sites.

And in the context of the importance of this process for starch biosynthesis, the usual comparative analyzes of starch content in leaves or seeds in wild and mutant plants are lacking. The process itself, as the authors themselves note, is very complex, and it is not known whether AGPase activity is the factor that limits the efficiency of the entire process.

My two concluding remarks do not detract from the value of the paper - perhaps the authors will use it in the future? The paper is ready for printing after minor editorial corrections

Author Response

Reviewer 1

Effect of phosphorylation sites mutations on the subcellular localization and activity of AGPase Bt2 subunit: Implications for improved starch biosynthesis in Maize. By Guowu et al

In the present manuscript, authors focus on effects of phosphorylation sites mutations on the subcellular localization and activity of AGPase Bt2 subunit.

I read this manuscript with interest.

Author response: We are grateful to the reviewer for their careful reading of our paper and their insightful comments and suggestions. We have considered all feedback and made the necessary adjustments accordingly. For ease of identification, all changes have been marked in red in the manuscript file.

Reviewer Comments to the Authors:

1. Please change the keywords, they are probably meant to help less savvy readers and basically only "starch synthehis" says anything, the others are incomprehensible or imprecise: "phosphorylation" of what???, subcellular localization of what?, what is Bt2 activity?

Author response: Thank you for pointing this out, there were mistakes. We have updated the keywords in the manuscript. Following changes have been made (Line 34).

Keywords: AGPase; Enzyme activity regulation; AGPase phosphorylation; Subcellular localization of AGPase

2. In the "Introduction", the authors describing the synthesis of starch should mention the role of starch phosphorylase (SP) [39], especially since they mention this enzyme in the discussion.

Author response: Thank you for pointing this out. We have updated the introduction portion and added with the information about starch phosphorylase (SP) enzyme role in the process of starch biosynthesis.

Following changes have been made (Lines 55-60).

In addition, there are indications that AGPase does not entirely involve in starch bio-synthesis as it requires a primer of reasonable chain length [8]. Based on biochemical evidence, it is assumed that starch phosphorylase (SP) involved in the initiation and amplification starch biosynthesis in plants [9, 10]. There can be two possible routes for starch synthesis, either through the PHO1 or AGPase/SS, which depend on the availa-bility of substrate-level (Glc-1-P) [11, 12].

3. It is worth mentioning that the publication is written very carefully and without typos, so in the section "Materials and methods" in chemical formulas numbers should be formatted with appropriate upper or lower indices, e.g., in Na2CO3 and other chemical formulas: 2.4, 2.6, 2.7, 2.8.

Author response: We are grateful to the reviewer for their careful reading of our paper pointing out these mistakes. We have carefully checked and updated the Materials and Methods section. The chemical formulas have been formatted appropriately.

Following changes have been made (Lines 162-242).

2.4. Subcellular Localization Analysis

Subcellular localization vectors were constructed for both wild type and mutant type, leading to the creation of 2300-Bt2-eGFP, 2300-Bt2-S10/T451/T462A-eGFP, and 2300-Bt2-S10/T451/T462E-eGFP fusion plasmids as per described by [29]. The selected restriction enzyme sites were KpnI and XbaI, with homologous recombination primers listed in Table 1. Constructed plasmids were introduced into endosperm protoplasts using the Polyethylene Glycol-Calcium (PEG-Ca²⁺) transformation method. Following transformation, protoplasts were cultured for 16 hours either in the dark or under low-light conditions. Post-culture, the intracellular distribution of GFP was examined using microscopy.

2.5. Yeast-two Hybrid Assay

The fusion plasmids pGADT7-Bt2, pGADT7-Bt2-S10/T451/T462A, pGADT7-Bt2- S10/T451/T462E, and pGBKT7-Sh2 were constructed according to [30]. The restriction enzyme sites used were EcoRI and BamHI, and the homologous recombination primers are presented in Table S1. The combination of pGADT7 and pGBKT7 was utilized as a negative control. The resulting plasmid combinations were introduced into Y2H yeast-competent cells using the PEG/LiAc method. Following the transformation process, the cells were spread onto selection media lacking tryptophan (SD/Trp). The plates were incubated at 28°C for approximately three days. Positive clones were then selected and cultured in liquid media without two necessary nutrients until reaching an optical density (OD600) between 0.5 and 0.8. The yeast culture was then collected, washed with sterile water, and spotted onto both two-deficient and four-deficient solid media at various concentration gradients (10^-1, 10^-2, 10^-3, 10^-4, from left to right). The plates were cultured for an additional 2-3 days and the growth of individual yeast colonies was subsequently observed.

2.6. Bimolecular Fluorescence Complementation

The BiFC expression vectors were constructed utilizing E2913 (pSAT6-nEYFP-N1) and E3086 (pSAT6-cEYFP-N1) to harbor the Bt2 (and its mutant forms) and Sh2 genes, respectively. This yielded fusion plasmids: E2913-Bt2-N YFP, E2913-Bt2-S10/T451/ T462A-N YFP, E2913-Bt2-S10/T451/T462E-N YFP, and E3086-Sh2-C YFP. The restriction enzyme sites selected for this process were EcoRI and SmaI, and the relevant homologous recombination primers are documented in Table S1. The combination of E2913-N YFP and E3086-C YFP was employed as a negative control. The combined plasmids were introduced into maize leaf etiolated seedling protoplasts using the PEG-Ca²⁺ transformation method. Following a 24-hour incubation period in darkness, the YFP yellow fluorescence signal was inspected under a confocal microscope.

2.7. Protoplasm Preparation

Seedling stage leaves exhibiting yellow heads was delicately sliced into 0.5-1 mm wide leaf fillets using a sharp blade. Concurrently, endosperm from maize (7-9 DAP) was carefully removed with precision tweezers and swiftly segmented into minute pieces. To prevent desiccation, these were immersed in an enzyme solution composed of 1.5% cellulase, 0.75% pectin, 10 mM MES, 600 mM mannitol, 10 mM CaCl₂, and 0.1% BSA. The endosperm underwent enzymatic hydrolysis for 4-6 hours. Post-hydrolysis, an equal volume of W5 solution (154mM NaCl, 125mM CaCl₂, 5mM KCl, 2mM MES-KOH) was added, followed by brief centrifugation at 90g for 3 minutes at 4°C. The supernatant was discarded, protoplasts were resuspended in 5mL of W5, and the centrifugation step was repeated. The supernatant was subsequently discarded, 1mL of W5 was added, and the solution was allowed to rest on ice for 30 minutes. The protoplasts were then resuspended in MMG solution (400mM mannitol, 15mM MgCl₂, 4mM MES-KOH, pH 5.6) at a density of approximately 2×10^5 protoplasts/mL. The transformation process was initiated by adding 10-20μg of plant expression vector to the protoplast suspension. A gentle mixing followed, succeeded by the addition of 100μL of PEG-Ca²⁺ solution (4M mannitol, 10mM CaCl₂, 40% PEG). The induced transformation was carried out in the darkness for 15 minutes, then halted by adding 400 μL of W5 solution. Following a brief centrifugation at 90g for 2 minutes at room temperature and discarding the supernatant, an additional 100μL of W5 solution was gently mixed in. The protoplasts were incubated in the dark at 28°C for 12 hours and subsequently observed under a microscope. Protoplasts were then gathered via a brief centrifugation at room temperature and a fraction of this was selected for fluorescence signal observation under a confocal microscope.

4. Line 68:  instead of "ADPG transporter" should be ADP-Glc transporter

Author response: Sorry it was a mistake; we have replaced the word "ADPG transporter" with “ADP-Glc transporter”.

Following changes have been made (Lines 76-78).

These findings suggest a unique pathway in cereal endosperm starch synthesis that requires an ADP-Glc transporter to shuttle ADP-Glc, generated by cytoplasmic AGPase, into the plastids [17, 18].

5. Line 375: instead of [40] should be [39]

Author response: Sorry it was a mistake; we have updated the citations and numbers have been updated in whole manuscript.

6. Overall, I find the paper very interesting and relevant for a better understanding of AGPase activation. If there were other aspects that I think would improve the significance of the work, they would be bioinformatics analyzes and modeling of AGPase subunit structures as a function of sequence and possibly phosphorylation of the phosphorylation sites.

And in the context of the importance of this process for starch biosynthesis, the usual comparative analyzes of starch content in leaves or seeds in wild and mutant plants are lacking. The process itself, as the authors themselves note, is very complex, and it is not known whether AGPase activity is the factor that limits the efficiency of the entire process.

My two concluding remarks do not detract from the value of the paper - perhaps the authors will use it in the future? The paper is ready for printing after minor editorial corrections.

Author response: First and foremost, we appreciate your time and effort in reviewing our paper. We are glad to hear that you found the content interesting and relevant for understanding AGPase activation.

Thank you for the constructive feedback. We wholeheartedly agree with your suggestion on the significance of bioinformatics analyses and modeling of AGPase subunit structures. Taking into account sequence variations and the effect of phosphorylation on these sites would indeed bring deeper insights. Furthermore, the comparative analyses of starch content in leaves or seeds in both wild and mutant plants is a valid point that would complement our current study. We acknowledge that the intricacies of the process have layers that we have yet to delve deep into.

To address your concerns, this study is intended as a preliminary exploration into the significance of phosphorylation on AGPase. We are currently conducting further experiments to ascertain and corroborate our findings. The suggestions you've mentioned are certainly on our radar, and we hope to incorporate them in our subsequent studies to paint a comprehensive picture.

Once again, thank you for your invaluable suggestions and insights. We will attend to the editorial corrections and ensure the paper is refined for printing.

We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may have.

Thanks!

Author Response File: Author Response.pdf

Reviewer 2 Report

 

In this manuscript titled: Effect of Phosphorylation Sites Mutations on AGPase Bt2 Subunit: Implications for Maize Starch Biosynthesis'' the authors demonstrated the advanced agricultural sustainability hinges on unraveling the nuances of starch biosynthesis, a pivotal process in crop yield optimization. ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme catalyzing a key step in starch production. This study focuses on phosphorylation-mediated AGPase regulation as a potential avenue for augmenting starch synthesis in maize. By introducing point mutations at phosphorylation sites within AGPase Bt2 subunit, the investigation probes alterations in both subcellular localization and enzymatic activity. Notably, while subcellular positioning remains unchanged, heightened AGPase activity emerges from simulated phosphorylation mutations, promising enhanced ADP-Glc production—a central starch precursor. While shedding light on these vital connections, certain considerations warrant attention. Expanding mechanistic insights into mutation-driven activity changes and discerning wider metabolic implications could further amplify the study's implications. Additionally, integrating experimental validation and a holistic functional exploration of AGPase's roles could amplify the manuscript's relevance in enhancing crop improvement and biotechnology.

Comments:

1.       Could you briefly explain why AGPase is considered a rate-limiting factor in starch production and its significance for crop yield and agricultural applications?

2.       Could you provide a brief overview of how phosphorylation is believed to influence AGPase activity and starch biosynthesis?

3.       Could you elaborate on the significance of iTRAQTM in identifying these sites and its potential implications for understanding AGPase regulation?

4.       What is the rationale behind focusing on these specific aspects, and how might alterations in localization and interaction impact starch biosynthesis?

5.       Could you delve into the mechanistic details of how specific mutations affect enzyme activity? Are there any insights into the downstream consequences of altered AGPase activity on starch synthesis?

6.       How does this align with previous research, if any, and what might be the underlying reasons for the lack of impact on these aspects?

7.       How does an increase in AGPase activity, as observed in simulated phosphorylation mutations, directly translate to improved starch biosynthesis? Could you elaborate on the connection between increased AGPase activity and enhanced starch production?

8.       What potential benefits might arise from enhanced starch production in maize, and how might this finding contribute to addressing agricultural challenges or improving crop yield?

9.       Considering maize's importance as a crop, do you foresee potential applications or implications of these findings for other crop species that undergo starch biosynthesis? How might this research contribute to broader agricultural contexts?

10.   How were the simulated phosphorylation point mutations generated, and were there any experimental validations of the observed effects on AGPase activity? How might the findings be further substantiated through additional experimental approaches?

11. Minor spell check and grammar corrections are required.

 

Minor spell check and grammar corrections are required.

Author Response

Reviewer 2

In this manuscript titled: Effect of Phosphorylation Sites Mutations on AGPase Bt2 Subunit: Implications for Maize Starch Biosynthesis'' the authors demonstrated the advanced agricultural sustainability hinges on unraveling the nuances of starch biosynthesis, a pivotal process in crop yield optimization. ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme catalyzing a key step in starch production. This study focuses on phosphorylation-mediated AGPase regulation as a potential avenue for augmenting starch synthesis in maize. By introducing point mutations at phosphorylation sites within AGPase Bt2 subunit, the investigation probes alterations in both subcellular localization and enzymatic activity. Notably, while subcellular positioning remains unchanged, heightened AGPase activity emerges from simulated phosphorylation mutations, promising enhanced ADP-Glc production—a central starch precursor. While shedding light on these vital connections, certain considerations warrant attention. Expanding mechanistic insights into mutation-driven activity changes and discerning wider metabolic implications could further amplify the study's implications. Additionally, integrating experimental validation and a holistic functional exploration of AGPase's roles could amplify the manuscript's relevance in enhancing crop improvement and biotechnology.

Author response: We are grateful to the reviewer for their careful reading of our paper and their insightful comments. We have considered all feedback and made the necessary adjustments accordingly. For ease of identification, all changes have been marked in red in the manuscript file.

Reviewer Comments to the Authors:

1. Could you briefly explain why AGPase is considered a rate-limiting factor in starch production and its significance for crop yield and agricultural applications?

Author response: Thank you for quarry. A rate-limiting step or factor in a process is the slowest step, and it dictates the overall rate of that process. In the context of starch biosynthesis, AGPase catalyzes the conversion of ATP and Glucose-1-Phosphate (Glc-1-P) to adenosine diphosphate glucose (ADP-Glc). This reaction is considered the first committed step in starch synthesis. Since this is an early step and its product, ADP-Glc, is the primary substrate for the subsequent starch production (act as a substrate to extend the α-1,4 linked glucans by Starch Synthase (SS)), the rate at which this reaction occurs can effectively control the overall rate of starch synthesis.

Significance for Crop Yield and Agricultural Applications:

1. Starch Content:Starch is a primary storage carbohydrate in many crops, especially cereals like rice, maize, and wheat. The amount of starch in seeds directly affects the seed's nutritional quality and yield.
2. Energy Reserve:Starch serves as an energy reserve for plants. A plant's ability to store energy in the form of starch can determine its survival during periods of low light or other stress conditions. This is especially crucial for crop plants that need to ensure seed production.
3. Potential for Genetic Modification:By understanding and potentially manipulating AGPase activity, researchers might increase the starch content in crops. Such genetic modifications could lead to crop varieties that produce higher yields or have enhanced nutritional profiles.
4. Economic and Sustainability Implications:Starch is not just a food source. It's also used in various industries like biofuel production, textiles, and paper. Boosting starch production in crops could have wide-reaching economic and sustainability implications, given the demand for sustainable biofuels and other starch-derived products.

In general, AGPase role as a rate-limiting enzyme in starch biosynthesis makes it a critical point of interest for researchers aiming to enhance crop yields and improve the efficiency of starch production for both food and industrial applications.

Following changes are also incorporated in the manuscript (Lines 45-55).

The complex process of starch biosynthesis fundamentally relies on a sequence of cat-alytic enzymes, including adenosine diphosphate glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE), and starch debranching enzyme (DBE) [6]. Of these, AGPase holds a crucial position as a rate-determining enzyme in starch biosynthesis, catalyzes the conversion of ATP and Glu-cose-1-Phosphate (Glc-1-P) to adenosine diphosphate glucose (ADP-Glc). This reaction is considered the first committed step in starch synthesis. Since this is an early step and its product, ADP-Glc, is the primary substrate for the subsequent starch production (act as a substrate to extend the α-1,4 linked glucans by SS [7], the rate at which this reaction occurs can effectively control the overall rate of starch synthesis.

2. Could you provide a brief overview of how phosphorylation is believed to influence AGPase activity and starch biosynthesis?

Author response: Thank you for quarry. In the context of AGPase and starch biosynthesis, phosphorylation plays a crucial regulatory role.

1. Activation of AGPase:In many plants, the activity of AGPase is enhanced by the phosphorylation of its subunits. When the enzyme is phosphorylated, it can have a higher affinity for its substrates, and the overall reaction rate can increase.
2. Modulation of Protein Interactions:Phosphorylation can also influence the interaction between the small and large subunits of AGPase, potentially affecting its overall structure and activity.
3. Regulation by Other Effectors:The effect of other allosteric effectors on AGPase (like 3-phosphoglycerate and inorganic phosphate) can also be modulated by its phosphorylation state.

Given that AGPase is a rate-limiting enzyme in starch biosynthesis, its activity is directly correlated with the rate of starch production. So, when phosphorylation enhances AGPase activity:

1. Increased ADP-Glc Production:Since AGPase is responsible for producing ADP-Glc, the primary substrate for starch synthesis, increased enzyme activity translates to more ADP-Glc availability for starch production.
2. Higher Starch Content:Since the early product of AGPase is ADP-Glc, which is the primary substrate for the subsequent starch production. It may have concluded that enhanced AGPase activity can lead to increased starch synthesis.
3. Feedback Regulation:Starch biosynthesis is not an isolated pathway; it interacts with other metabolic pathways in the cell. The products and intermediates of starch synthesis can serve as signals for other processes. An enhanced rate of starch synthesis due to AGPase activation might, therefore, have broader implications on plant metabolism.

In general, phosphorylation acts as a regulatory switch for AGPase activity. By modulating its activity, phosphorylation can directly influence the rate of starch synthesis, making it a key player in the overall carbohydrate metabolism of plants.

Following changes are also incorporated in the manuscript (Lines 79-95).

The regulation of AGPase, which varies across tissues and cells, profoundly influences its activity. Its function is co-modulated via diverse regulatory mechanisms, encom-passing redox [19], allosteric [20], and transcriptional approaches [21, 22]. With the progression of experimental methodologies, notably mass spectrometry, and affinity chromatography, comprehension of proteomes and phosphoproteomes has been significantly extended. Previous research has identified phosphorylated AGPase in a variety of species and tissues [23, 24]. In particular, grain-focused studies have indicated the occurrence of AGPase subunit phosphorylation. For instance, differential gel electrophoresis analysis of grown wheat seeds inferred that the small subunit of plastid AGPase undergoes phosphorylation, although the specific site of phosphorylation remained unidentified [25]. Past experiments in our laboratory suggest that when samples underwent alkaline phosphatase treatment for gel activity measurement, the resulting enzymatic activity was less compared to the phosphorylated control, insinuating an interrelationship between AGPase activity and phosphorylation [26]. More recently, research has verified the phosphorylation of the AGPase subunit in the wheat endosperm [27], with in vitro trials involving recombinant AGPase from wheat endo-sperm and calcium-dependent protein kinase indicating that phosphorylation emerges in the either of subunits.

3. Could you elaborate on the significance of iTRAQTM in identifying these sites and its potential implications for understanding AGPase regulation?

Author response: Thank you for quarry. iTRAQ™ serves as a sophisticated tool in the realm of proteomics, offering a comprehensive and quantitative approach to understanding protein modifications. One of the key advantages of iTRAQ™ is its ability to simultaneously analyze multiple samples in a single experiment. This is crucial for efficient comparison of phosphorylated versus non-phosphorylated protein states across different treatments or conditions. iTRAQ™ labels peptides with isobaric tags, allowing for precise quantitative comparisons. This means that not only can you detect a phosphorylation event, but you can also determine the relative extent of that phosphorylation under various conditions. iTRAQ™ enhances the detection of low-abundance peptides. This heightened sensitivity is crucial when detecting post-translational modifications, which might occur only under specific conditions or at particular developmental stages.

Its application in deciphering the phosphorylation dynamics of AGPase is instrumental in broadening our understanding of starch biosynthesis regulation, with implications for crop science and agriculture. Using iTRAQ™, researchers can pinpoint the exact sites on AGPase (or any protein) that are phosphorylated. This detailed knowledge is crucial for understanding how phosphorylation affects the protein's function. By comparing the relative abundances of phosphorylated AGPase peptides under different conditions (e.g., in the presence or absence of certain kinases), researchers can gain insights into the potential mechanisms or pathways that activate or deactivate AGPase. Once the phosphorylation sites are identified, they become targets for further investigations, such as mutagenesis studies. By creating mutants where these sites are altered, researchers can then explore the functional significance of each phosphorylation event in AGPase regulation.

Further details can be found in Yu et al., 2019 as we had conducted this experiment and Mass Spectrum data is shown in that publication.

Following changes are also incorporated in the manuscript (Lines 121-127).

2.2 iTRAQ^TM Labeling and Mass Spectrometry Analysis

The iTRAQ^TM method [28] was utilized to identify phosphorylated proteins in this study. Three samples of Mo17 corn 25 DAP grain protein were collected during both daytime and night, and the experiment was repeated. To begin, sample lysis and protein extraction were performed using an SDT buffer consisting of 4% SDS, 100 mM Tris-HCl, and 1 mM DTT at pH 7.6. The concentration of the extracted proteins was determined using the BCA Protein Assay Kit from Bio-Rad, USA.

4. What is the rationale behind focusing on these specific aspects, and how might alterations in localization and interaction impact starch biosynthesis?

Author response: Thank you for quarry. In our prior study, we hypothesized that AGPase undergoes phosphorylation and can affect these parameters. This was subsequently confirmed through the Phos Tag enrichment experiment as described by Yu et al., 2019. We identified multiple phosphorylation sites on AGPase. Building on this discovery, we further investigated and characterized the functional roles of these specific phosphorylation sites on AGPase.

Furthermore, in relation to localization, the specific aim was to confirm the localization is not to be effected by site directed mutations as starch biosynthesis in plants hinges on the precise localization and interaction of key enzymes, such as AGPase. Proper subcellular localization ensures that enzymes are positioned to access their necessary substrates, with starch predominantly being synthesized in specialized compartments like plastids. The interactions between enzymes, particularly the formation of multi-subunit complexes, are crucial for optimal enzyme activity and regulatory control. Any deviations in enzyme localization or interaction can disrupt starch synthesis, potentially affecting plant energy reserves, growth patterns, and overall crop yields.

5. Could you delve into the mechanistic details of how specific mutations affect enzyme activity? Are there any insights into the downstream consequences of altered AGPase activity on starch synthesis?

Author response: Thank you for quarry. Specific mutations in enzymes can dramatically influence their activity, often by reshaping their active sites, interfering with regulatory molecule binding, disrupting interactions between subunits, or modifying stability and post-translational modifications. When we focus on AGPase, a critical player in starch synthesis, altered activity can have cascading effects on the entire starch synthesis pathway. Any change in the rate of ADP-Glc production, a direct outcome of AGPase's function, impacts the structure, amount, and quality of starch produced. These modifications not only affect the plant's energy management but can also influence crop yields and their subsequent applications. Additionally, the interconnected nature of metabolic pathways means that changes in AGPase activity can ripple through related pathways, invoking feedback mechanisms and potentially affecting other vital plant processes.

To address your concerns, this study is intended as a preliminary exploration into the significance of phosphorylation on AGPase. We are currently conducting further experiments to ascertain and corroborate our findings.

Following changes are also incorporated in the manuscript (Lines 410-433).

5. Conclusions

In this study, the point mutation of Bt2, the small subunit, was found not to have an impact on its subcellular location. Bt2 remained localized within the nucleus and the cytoplasm, suggesting that the fundamental cellular distribution of the protein was not perturbed by these point mutations. Furthermore, this point mutation did not appear to hinder Bt2 interaction with its larger counterpart, Sh2. In maize leaf protoplasts, their interaction continued to take place primarily within the chloroplast, maintaining the subcellular site for this crucial protein-protein interaction. Intriguingly, the point mutations of Bt2 had a substantial effect on the activity of the AGPase enzyme, particularly in the context of its phosphorylation state. When the mutated Bt2 was subject to phosphorylation modifications, the activity of AGPase increased, which hints that phosphorylation at specified site could enhance starch synthesis process. Conversely, dephosphorylation led to a decrease in AGPase activity, implying that phosphorylation could be a vital regulatory mechanism for the functionality of this enzyme. The nuanced influence of these point mutations on AGPase activity, depending on its phosphorylation status, suggests a critical role for phosphorylation in modulating the enzymatic activity of AGPase. This dynamic could be harnessed to optimize starch synthesis in crop production, an area that warrants further investigation. Through the creation and examination of mutants with augmented AGPase activity, this research sets the stage for the possible creation of crop variations with superior starch content. Such advancements could enhance both crop yield and quality. Additionally, comprehension of the way the phosphorylation status of Sh2 affects AGPase activity may lead to the cultivation of crop variations with altered starch characteristics, potentially amplifying their utility across various industries, such as the food, pharmaceutical, and biofuel sectors.

6. How does this align with previous research, if any, and what might be the underlying reasons for the lack of impact on these aspects?

Author response: The research delves into the pivotal role of AGPase and its potential regulation via phosphorylation in maize. It builds upon previous studies that hinted at AGPase's phosphorylation-based regulation Yu et al., 2019. This study adds depth by revealing that while point mutations at phosphorylation sites alter AGPase activity, they don't affect its subcellular localization or the interaction dynamics between its subunits. The unchanged localization and interactions might stem from the inherent stability of the protein's structure, potential compensatory mechanisms within the protein, and undisturbed intrinsic localization signals and interaction domains. Thus, this research not only aligns with prior findings but paints a more intricate picture of AGPase regulation. The results underscore the potential of targeted genetic interventions for enhancing maize starch production without compromising key cellular processes.

Following changes are also incorporated in the manuscript (Lines 79-95).

The regulation of AGPase, which varies across tissues and cells, profoundly in-fluences its activity. Its function is co-modulated via diverse regulatory mechanisms, encompassing redox [19], allosteric [20], and transcriptional approaches [21, 22]. With the progression of experimental methodologies, notably mass spectrometry, and affinity chromatography, comprehension of proteomes and phosphoproteomes has been significantly extended. Previous research has identified phosphorylated AGPase in a variety of species and tissues [23, 24]. In particular, grain-focused studies have indicated the occurrence of AGPase subunit phosphorylation. For instance, differential gel electrophoresis analysis of grown wheat seeds inferred that the small subunit of plastid AGPase undergoes phosphorylation, although the specific site of phosphorylation remained unidentified [25]. Past experiments in our laboratory suggest that when samples underwent alkaline phosphatase treatment for gel activity measurement, the resulting enzymatic activity was less compared to the phosphorylated control, insinuating an interrelationship between AGPase activity and phosphorylation [26]. More recently, research has verified the phosphorylation of the AGPase subunit in the wheat endosperm [27], with in vitro trials involving recombinant AGPase from wheat endo-sperm and calcium-dependent protein kinase indicating that phosphorylation emerges in the either of subunits.

7. How does an increase in AGPase activity, as observed in simulated phosphorylation mutations, directly translate to improved starch biosynthesis? Could you elaborate on the connection between increased AGPase activity and enhanced starch production?

Author response: Thank you for underlining this. We have conducted the enzyme assay to check the effect of phosphorylation on AGPase activity by using recombinant AGPase which suggests an increase in activity (Results, Lines 314-328). On the bases of this, we have speculated that phosphorylation can increase the AGPase activity. In the context of starch biosynthesis, AGPase catalyzes the conversion of ATP and Glc-1-P to ADP-Glc. This reaction is considered the first committed step in starch synthesis. Since this is an early step and its product, ADP-Glc, is the primary substrate for the subsequent starch production (act as a substrate to extend the α-1,4 linked glucans by SS), the rate at which this reaction occurs can effectively control the overall rate of starch synthesis. There were some mistakes or we failed to express our views clearly but now conclusion section has been updated and we are hoping that it is more robust and well-written (Lines 418-420).

Following changes are also incorporated in the manuscript (Lines 314-328).

3.6. Effects of Site-Directed Point Mutations on AGPase Activity

UG221-Sh2, along with various mutant types of UG221-Bt2, were co-transfected into the protoplasts of etiolated seedlings for transient expression. The transfected protoplasts were then cultivated in darkness for 16 hours. Subsequently, ATP levels were evaluated using the ATP detection kit provided by Beyotime Biotech, with the experimental findings illustrated in Figure 5. Taking the Bt2+Sh2 interaction as a base-line, a significant decrease in AGPase activity was observed in the Bt2-S10/T451/T462A+Sh2 measurements. Conversely, the AGPase activity assessed by Bt2-S10/T451/T462E+Sh2 demonstrated an increase. These results provide compelling evidence that point mutations can indeed alter AGPase activity. In this experiment, it was observed that the simulation of dephosphorylation led to a reduction in AGPase activity, whereas the simulation of phosphorylation prompted an increase in AGPase activity. This underscores the profound impact of phosphorylation status on enzymatic function, highlighting the intricate relationship between post-translational modifications and enzyme activity.

(Lines 418-420)

When the mutated Bt2 was subject to phosphorylation modifications, the activity of AGPase increased, which hints that phosphorylation at specified site could enhance starch synthesis process.

8. What potential benefits might arise from enhanced starch production in maize, and how might this finding contribute to addressing agricultural challenges or improving crop yield?

Author response: Augmented starch biosynthesis in maize presents a significant opportunity for advancements in agronomic research. An increase in maize's starch content directly correlates with enhanced crop yield, facilitating a more abundant harvest per unit area and augmenting its nutritional parameters to address the dietary demands of increasing global populations. Beyond merely serving as a pillar for food security, this enhancement has substantial economic implications, potentially increasing profit margins for farmers and bolstering regions predominantly dependent on maize as a primary food source. Concurrently, elevated maize starch levels can be instrumental for the biofuel sector, positioning maize as an efficient, renewable substrate for ethanol production. This ascendancy also augments its value for industries utilizing starch for diverse applications, ranging from bioplastics synthesis to textile manufacturing. A particularly salient aspect of this enhancement could manifest in climatic resilience. Starch-rich maize may exhibit augmented resistance to variable environmental stressors, mitigating the impacts of erratic climatic events. Delving deeper into the mechanisms of starch biosynthesis potentially unveils opportunities for genetic modifications, facilitating the development of maize cultivars optimized for distinct challenges, from drought tolerance to specific industrial requisites. In essence, advancements in maize starch biosynthesis hold promise for addressing both current agronomic challenges and fostering sustainable agricultural practices.

9. Considering maize's importance as a crop, do you foresee potential applications or implications of these findings for other crop species that undergo starch biosynthesis? How might this research contribute to broader agricultural contexts?

Author response: The findings on maize starch biosynthesis have broader implications beyond just maize. Many plants share fundamental principles of starch biosynthesis, so insights from maize's AGPase regulation might be applied to staple crops like rice, wheat, and potato. If altering maize AGPase can boost starch production without adverse effects, similar genetic modifications could be explored in other crops, leading to higher starch yields beneficial for both food and industry. Additionally, crops with enhanced starch content might better withstand unpredictable climate conditions, ensuring stable yields. Overall, while maize is the primary focus, the research could influence wider crop research, potentially revolutionizing various aspects of agriculture.

10. How were the simulated phosphorylation point mutations generated, and were there any experimental validations of the observed effects on AGPase activity? How might the findings be further substantiated through additional experimental approaches?

Author response: Simulated phosphorylation point mutations were typically generated using site-directed mutagenesis, wherein specific nucleotide sequences in the DNA encoding target amino acids are altered, leading to the substitution of these amino acids to mimic the effects of phosphorylation (Materials and Methods, Lines 148-161; Results, Lines 251-257).

To validate the observed effects on AGPase activity, enzymatic assays have been employed. Such assays measured the conversion rates of substrates, in this case, ATP and Glucose-1-Phosphate, to the product ADP-Glc, comparing the activity of the wild type and mutant enzymes (Materials and Methods, Lines 219-242; Results, Lines 322-328).

For further substantiation, multiple experimental approaches could be undertaken. Proteomic analyses can confirm whether the mutations truly mimic phosphorylation. Structural studies, like X-ray crystallography, can elucidate conformational changes brought about by these mutations. Additionally, examining the phenotype of plants carrying these mutations, in terms of growth, starch content, and yield, would provide a holistic understanding of the mutation's impact in a real-world context. In summary, while initial findings from simulated mutations provide compelling insights, a suite of complementary experimental approaches can enrich and solidify our understanding of AGPase's regulatory nuances.

Following changes are also incorporated in the manuscript (Lines 148-161).

2.3. Site-Directed Point Mutations

Site-specific primers for gene mutations Bt2-S10A-F/R, Bt2-S10E-F/R, Bt2-T451A-F/R, Bt2-T451E-F/R, Bt2-T462A-F/R, and Bt2-T462E-F/R were designed ac-cording to NCBI guidelines, leading to a total of six pairs (primer sequences detailed in Table S1).

(Lines 251-257)

3.2 Site-Directed Point Mutations

The Bt2 gene spans a total length of 1428 base pairs (bp). Specific mutation primers were engineered and the resulting sequences aligned using DNAman software. Figure 1 presents the sequence outcomes of the mutation sites following simulated processes of phosphorylation (E) and dephosphorylation (A). These confirmed mutated clones were then used for amplification of the mutated Bt2, paving the way for future recombination with required vectors.

 

 

 

Figure 1. Sequence diagram of site-directed point mutations. W-Bt2 represents the reference Bt2 sequence from the NCBI database. Bt2-S10A/T451A/T462E highlights the sequence region en-compassing the serine 10 site replaced with alanine, threonine 451 sites replaced with alanine, and threonine 462 sites replaced with alanine. Bt2-S10E/T451E/T462E highlights the sequence region encompassing the serine 10 site replaced with glutamic acid, threonine 451 sites replaced with glutamic acid, and threonine 462 sites replaced with glutamic acid. White sequence lines demonstrate the primers used for site-directed mutagenesis.

(Lines 219-242)

2.8. Determination of AGPase Activity

To assess the activity of AGPase the change in RLUC (Renilla luciferase) value corresponding to ATP production was measured using the ADP/ATP luminescence detection kit (Beyotime). Fusion plasmids 221-Bt2, 221-Bt2-S10/T451/T462A, 221-Bt2-S10/T451/T462E, and 221-Sh2 were constructed utilizing BamHI and SacI re-striction sites, with the corresponding homologous recombination primers delineated in Table S1. AGPase activity was inferred based on the enzyme's reversible property that facilitates ADPG catalysis to consume Pi and generate ATP. The resultant ATP volume was quantified to assess AGPase activity changes. These constructed plasmids were introduced into the etiolated seedling protoplasts via PEG-Ca2+ transformation. Following 24 hours of dark cultivation, the ATP levels were determined. For in vitro AGPase activity assessment, an ATP detection kit was employed. After 14-16 hours of dark incubation, the protoplasts were harvested via centrifugation at room tempera-ture (90g, 1 minute). RLUC activity was evaluated using the Luciferase Assay System (Promega), adhering to the instructions outlined by the ATP Assay Kit (Beyotime). GUS activity was assessed by adding 40μL of cell lysate supernatant to 40μL of 4-methylumbelliferyl-β-D-glucuronide (MUG) reaction substrate. After thorough mix-ing, 30μL of the reaction mixture was halted with 60μL of 0.3M Na2CO3, and GUS ac-tivity was immediately recorded at time zero after sampling 75μL of the mixture. The remaining 50μL of the reaction solution was further incubated in the dark at 37°C for 4 hours, then halted with 60μL of 0.3 M Na2CO3. GUS activity was recorded after the complete 4-hour incubation period. The halted reaction solution was transferred to a black 96-well cell culture plate, and GUS activity was assessed using a fluorescence spectrophotometer. The ratio of RLUC activity to GUS activity was used as a measure of AGPase activity.

(Lines 322-328)

These results provide compelling evidence that point mutations can indeed alter AGPase activity. In this experiment, it was observed that the simulation of dephosphorylation led to a reduction in AGPase activity, whereas the simulation of phosphorylation prompted an increase in AGPase activity. This underscores the profound impact of phosphorylation status on enzymatic function, highlighting the intricate relationship between post-translational modifications and enzyme activity.

11. Minor spell check and grammar corrections are required.

Author response: Thank you for your careful readings and recommendations. We have made significant improvements to the language of our manuscript, and it has been thoroughly reviewed by a native English speaker. Furthermore, grammar mistakes have also been checked and corrected. We trust that the quality of the language now meets the necessary standards, and we anticipate that it will not present any further issues.

 

We look forward to hearing from you regarding our submission. We would be glad to respond to any further questions and comments that you may have.

Thanks!

Author Response File: Author Response.pdf