From Plant to Paddy—How Rice Root Iron Plaque Can Affect the Paddy Field Iron Cycling
Round 1
Reviewer 1 Report
Maisch and co-authors present a study of how rice plant radial oxygen loss (ROL) can have impacts on rice paddy iron cycling outside of the impacts of iron-oxidizing bacteria and how rice plant iron oxide root plaques may be bioavailable to iron-reducing bacteria, specifically a consortia that is 95% Geobacter spp. The combined use of microelectrodes, plants, and high resolution imaging is a strength of this paper and the results will be of interest to the general readership of Soil Systems as well as a variety of scientists in the research field. A few minor revisions to strengthen the discussion and increase it’s impact on the field is discussed below.
The overall argument(s) of the paper could be strengthened by integrating a more wholistic view into the discussion of the paper. Specifically, in the discussion of the contribution of microaerophilic iron-oxidizing bacteria it could be of benefit to consider how microbial O2 sequestration could reduce the (micro)oxic zone surrounding the root surface, which could impact the ability of iron-reducing bacteria by increasing the possible active zone. The calculations included in this section also seem to overlook the possibility that some of the plant plaques formed via ROL may have instead occurred as a by-product of microbial metabolism if those microbes were present to sequester the oxygen. A range of possible percent iron oxide formation would perhaps have been more representative of the current uncertainty left after this study.
There is a lot of variability between oxygen production rates and iron plaque formation data. This is expected with environmental data, but can you elaborate on how this variability (so your high and low ends of these ranges) may affect your calculations in the discussion?
What are the biological implications of the different iron minerals that are formed? Please include this in the discussion.
In a suggestion of further study, it could be worth mentioning that a future study should include a similar set up comparing the iron plaquing mineral make-up from ROL vs. ROL + FeOB. This could potentially get at one of the lingering questions from this study and could potentially impact the conclusion that rice roots in isolation “heavily” impact biogeochemical cycling as this study is notably missing key members of iron cycling in rice paddies.
Further suggestions for point clarification and data representation are below.
Line 84: Can you elaborate on what was used to grow the plants under anoxic conditions with temperature and humidity control that was also light-protected? I wouldn’t know how to readily replicate this procedure. (Maybe in supplemental.)
Line 85: Perhaps rephrase to sterile 50% Hoagland solution, as it is phrased this way on line 92.
Lines 112-119: Is it necessary to define both M and Airon plaque if they are the same thing? Perhaps better to go with convention (don’t think there is one) or only use one variable. I would also move the equation for Aimage to its own line following its introduction on line 110.
Line 132: Should PFA be spelled out?
Lines 133 & 140: How long were these incubations?
Line 135: This sentence is confusing, presumably roots covered in plaque were sampled from a 52 day old plant, but the way it reads it sounds like you’re dunking them in root plaque.
Line 159: DAT is introduced here, but not defined until page 5.
Line 159: I don’t know that whiff is the best word, maybe “intermittent oxygen was detected”
Line 198 / Figure 3: As you astutely pointed out, both the average total root surface area and the surface area of root iron plaque increased, but I think it would be more informative to normalize the amount of root iron plaque increase by the amount of total surface area available at the different measuring points and could warrant moving the supplemental figure (S1B) to the paper itself. Figure 3A is somewhat misleading at a cursory glance since it makes it seem that the overall root coverage is increasing over time, while S1B shows that the average root covering is proportionally more consistent.
Figure 5: At which incubation time point was the photo for panel A taken? (e.g. after 43 hours incubation)
Figure 6: Include in figure legend for 6F which timepoint measurements are for (day 10).
Line 287: Improper use of the word “amount,” try “the large number of iron minerals…”
Figure 7: Similar concerns with the inset graph that is from Figure 3. Also, despite having talked about them earlier you are missing, for example, microaerophilic FeOB in your summary of the rhizosphere iron cycle. If you want to only show data from this study, consider rephrasing the figure title. This could also address my other concern, that the figure implies that only Geobacter spp. are involved in iron reduction in the plant rhizosphere. You may also consider moving Figure 7 to before the introduction of microaerophilic FeOB in the discussion altogether, as that would make the omission less glaringly obvious.
Line 479/480: “… would find their ideal niches conditions, even enhance Fe(II) oxidation…” needs rephrasing. Not entirely sure what the connotation of the statement was supposed to be.
Lines 310-338: The arsenic discussion is a bit over-reaching as no heavy metal studies were done here. Please streamline section and limit it to general implications that your data can make.
Line 487-488: Please re-phrase as your research did not specifically show this, but the sentence implies that it is based on the results of this study itself.
Author Response
Comments and suggestions for Authors from Reviewer 1
Maisch and co-authors present a study of how rice plant radial oxygen loss (ROL) can have impacts on rice paddy iron cycling outside of the impacts of iron-oxidizing bacteria and how rice plant iron oxide root plaques may be bioavailable to iron-reducing bacteria, specifically a consortia that is 95% Geobacter spp. The combined use of microelectrodes, plants, and high resolution imaging is a strength of this paper and the results will be of interest to the general readership of Soil Systems as well as a variety of scientists in the research field. A few minor revisions to strengthen the discussion and increase it’s impact on the field is discussed below.
The overall argument(s) of the paper could be strengthened by integrating a more wholistic view into the discussion of the paper. Specifically, in the discussion of the contribution of microaerophilic iron-oxidizing bacteria it could be of benefit to consider how microbial O2 sequestration could reduce the (micro)oxic zone surrounding the root surface, which could impact the ability of iron-reducing bacteria by increasing the possible active zone.
We appreciate the reviewer’s suggestion and idea for a more wholistic view onto microbial interactions at and around the rice roots. Moreover, we think interactions involving a narrow cycling of iron between Fe(III)-reducing and Fe(II)-oxidizing bacteria is absolute plausible.
In order to consider this assumption in the manuscript, we added a few sentences and raised this point in the discussion transitioning from microaerophilic Fe(II) oxidation to the impact of Fe(III)-reducing bacteria.
[L387-393]: “On the contrary, the sequestration of O2 from ROL by microaerophilic Fe(II)-oxidizing bacteria might diminish local O2 concentrations surrounding individual roots. Both, the production of ferric biominerals, as a product of microbial Fe(II) oxidation, and the depletion in O2 might create suitable conditions for Fe(III)-reducing microorganisms usually sensitive to O2. These bacteria could be attracted into the redox-active zone in the vicinity of the roots and conserve metabolic energy by microbially reducing the ferric minerals produced by microaerophilic Fe(II)-oxidizers. “
The calculations included in this section also seem to overlook the possibility that some of the plant plaques formed via ROL may have instead occurred as a by-product of microbial metabolism if those microbes were present to sequester the oxygen.
Unfortunately, we are not aware on which section the reviewer is giving the suggestion. However, we intentionally discussed the role of microaerophilic Fe(II)-oxidizing bacteria from L.347 on. In here, we calculated the amount of iron plaque precipitates formed by ROL only without the contribution of microaerophilic Fe(II)-oxidizing bacteria. Based on the findings reported in Maisch et al., 2019a and b, where the relative area in the rice plant rhizosphere and range of optimum conditions for microaerophilic Fe(II)-oxidizing bacteria was quantified, we estimated the impact and Fe(II) turnover rates considering reported cell numbers from literature for the estimates in the current study.
Based on results from Neubauer et al., 2007 who found an additional contribution of microaerophilic Fe(II)-oxidizing bacteria in total Fe(II) oxidation on roots of a wetland plant we concluded that the biotic impact adds up to the ROL-induced iron mineral formation as iron plaque. However, we agree that the microbial activity can sequester O2, stemming from ROL and potentially reduce the plant-induced iron mineral formation. We therefore added a statement into the corresponding paragraph.
[L372-379]: “Compared to the finding that more than 1.2 g soil-borne iron that can precipitate on rice roots within one cubic meter of flooded paddy soil per day, the impact of biogenic microaerophilic Fe(II) oxidation can vary to a large extent but has the potential to enhance net total iron mineral formation in the rhizosphere by approx. 3-30%. On the contrary, the microbial oxidation of soil-borne Fe(II) and the sequestration of O2 by these bacteria could potentially reduce abiotic oxidation kinetics and diminish the role of plant-mediated iron plaque formation suggesting that the net contribution to Fe(II) oxidation can be even more attributed to the microbial activity.”
A range of possible percent iron oxide formation would perhaps have been more representative of the current uncertainty left after this study.
We agree with the reviewer’s suggestion and considered the range in reported Fe(II) oxidation rates for microaerophilic Fe(II) oxidizing bacteria for calculating a range in microbially-formed biominerals calculated in the respective paragraph.
[L.364-372]: “…given the reported numbers of microaerophilic Fe(II)-oxidizing bacteria in wetland soils ranging from 7.1 x 102-1.1 x 106 g-1 dry weight soil (up to 4.1 x 105 cells cm-3 soil) [29] and reported Fe(II) oxidation rates for a large variety of microaerophilic Fe(II)-oxidizing bacteria ranging from 1.0-8.3∙10-16 mol Fe(II) cell-1 hour-1 [18, 33, 53, 54] net microaerophilic Fe(II) oxidation can conservatively be estimated to oxidize between 4.1 x 10-11 to 3.4 x 10-10 mol Fe(II) cm-3 (0.04 to 0.34 mmol Fe(II) m-3) wetland soil per hour regarding the highest reported cell numbers from paddy fields. This amount of microaerophilically induced iron mineral formation could result in a total of 0.03 to 0.4 g iron mineral precipitation within one m3 paddy soil per day under optimum conditions.”
There is a lot of variability between oxygen production rates and iron plaque formation data. This is expected with environmental data, but can you elaborate on how this variability (so your high and low ends of these ranges) may affect your calculations in the discussion?
We appreciate this suggestion and fully agree that variations in observed and reported root iron plaque formation should be included in the discussion on the mineral formation potential of rice plants. In the corresponding text section, we therefore explicitly pointed out when measured values were regarded as averaged and when a certain range of values considered for a quantitative calculation of iron mineral formation. Additionally, we re-phrased the main text and added quantitative estimates on ranges for ROL induced iron mineral formation.
[L.279-291]: “Here we found that an averaged total of approx. 75 cm2 per plant iron plaque surface area was calculated to have formed on the root surface until the end of the growth cycle after 45 DAT (Figure 2). It has been previously observed that root iron plaque can form on roots of rice plants and form a thick layer between 20-40 µm [41, 42]. Given the calculated iron plaque surface area and the range of reported iron plaque layer thickness, the resulting volume of iron plaque that can form on the roots until 45 DAT was calculated to reach values between 150 mm3 to 300 mm3 per plant. Since ferrihydrite was the most dominant iron mineral species identified in the iron plaque in our setups, the density of ferrihydrite of ρFh = 3.8 g/cm3 [43] was considered as an estimate for the density of all iron minerals that formed as root iron plaque. This estimate results in a total of 570 mg to 1,140 mg of ferrihydrite that can theoretically form on the root surface of one plant within 45 DAT. Considering that ferrihydrite consists of approx. 70 atomic weight % of iron atoms, this results in a net mass of around 400 mg to 800 mg iron that precipitated as iron minerals on the roots of each rice plant within only 45 days.”
What are the biological implications of the different iron minerals that are formed? Please include this in the discussion.
In the discussion about the crystallinity of root iron plaque minerals we discussed both the consequences for hydrogeological and biological processes and that [L.435-439] “changes in iron plaque mineralogy not only affect surface properties of the iron plaque itself but also decrease sorption capacities for e.g. nutrients and contaminants. A decrease in sorption capacities, as it was observed for higher crystalline iron minerals, can have drastic negative effects on contaminant retention [63-65].”
Additionally, we concluded for biological processes that [L.439-443] “the higher relative abundance of more crystalline iron plaque minerals was demonstrated to decrease the bioavailability for microbial Fe(III) reduction [66, 67] and thus the remobilization rate of iron plaque-derived Fe(II). We therefore conclude that, with time, iron plaque minerals are becoming more recalcitrant towards (abiogenic and microbial) Fe(III) reduction and that the microbial bioavailability correlates negatively with root age.”
Our hypothesis based on the observed differences in root iron plaque crystallinity was based on the observation that reported a [L.445-452] “significantly lower microbial Fe(III) reduction rate for Fe(III)-reducing bacteria grown on aged or higher-crystalline iron minerals compared to growth on freshly precipitated ferrihydrite. We therefore suspect that while the crystallinity of root iron plaque minerals increases with root age, the availability to serve as a substrate for Fe(III)-reducing bacteria gradually decreases during plant growth (Figure 2). Under these circumstances, we conclude that young roots (i.e. root tips) may represent a highly dynamic iron redox-hot spot. Here, freshly precipitated low-crystalline iron plaque minerals can form which immediately serve as ideal substrate for microbial Fe(III) reduction.”
We agree that the influence of changing mineralogical iron phases on the importance of biological processes was probably not clearly stated and therefore added an additional sentence to the main text.
[L.443-447]: “…In doing so, microbial processes, such as Fe(III) reduction and reductive dissolution might be inhibited by the abundance of more crystalline iron mineral phases shifting the dominance in impacting the iron plaque reduction to more prevalent abiotically-induced reducing processes, such as by humic acids or other plant-derived complexing compounds.”
In a suggestion of further study, it could be worth mentioning that a future study should include a similar set up comparing the iron plaquing mineral make-up from ROL vs. ROL + FeOB. This could potentially get at one of the lingering questions from this study and could potentially impact the conclusion that rice roots in isolation “heavily” impact biogeochemical cycling as this study is notably missing key members of iron cycling in rice paddies.
We are thankful for this suggestion and agree with the advice to include a statement that clearly emphasizes that reported results were obtained in a less complex analogue to an environmental rhizosphere system. Also, in terms of future studies, we agree that a suggestion was not clearly stated that could inspire and motivate readers to go that path. Therefore, we modified the main text substantially, added additional statements on the reduced complexity of the current system and added suggestions for future research performed in that field.
[L.525-547]: “These findings strongly support our hypothesis that rice roots can considerably impact the biogeochemical iron cycle in water-logged paddy fields. Not only the oxidative power of the plant root itself is catalyzing numerous iron redox processes but serves as an important conductor for O2 that spatio-dynamically enables and disables microbial iron redox reactions. Undoubtedly, the current study investigated rhizosphere processes on a rather simplified analogue to a more complex and interacting rice paddy ecosystem containing numerous other key members interacting with the rhizosphere trinity of plant, soil and bacteria. However, the current observations extent our understanding in the rhizosphere iron cycling and help to decipher individually that rice plant roots can be one of these key members for both the reductive and oxidative side of the soil-borne iron cycle. [14, 58]. We suggest future research to include similar approaches, increasing the integrity of biogeochemical interactions to fully decipher potential cross-links in the iron cycle between the enormous variety of participants. One potential step towards an understanding of the iron cycle in a more complex rhizosphere system could be the quantitative spatiotemporal investigation of root iron plaque formation incubated in the presence and absence of different members of iron-cycling bacteria, and the availability of soil-extracted soil organic matter or humic substances as metabolic substrate. Moreover, also different irrigation practices could be simulated in future studies. Soil redox conditions in the current setup were maintained constantly anoxic, representing water-logged paddy field. Periodic draining of paddy fields, however, might switch redox-conditions to prevalent oxic conditions, suppressing microbial Fe(III) reduction. Taken all these variable parameters together, the understanding of this spatio-dynamic small-scale iron redox rhizosphere system can have a huge impact on large scale observations and should be considered for future investigations of the rhizosphere iron redox cycle in paddy fields.”
Further suggestions for point clarification and data representation are below.
Line 84: Can you elaborate on what was used to grow the plants under anoxic conditions with temperature and humidity control that was also light-protected? I wouldn’t know how to readily replicate this procedure. (Maybe in supplemental.)
We appreciate the reviewer’s comment and agree that the information on experimental conditions is lacking in details. Besides referring to Maisch et al., 2019a in L.90, we added a paragraph into the Supporting Information describing the dimensions and preparation steps for setting up and incubating the plant growth containers:
[L.89] “…seedlings were transferred into anoxic and sterile rhizotrons (described in Maisch et al., 2019a and Supporting Information)…”
[in SI] “Plant growth containers.
Plant growth containers were made of transparent plexiglass (25 cm × 25 cm × 3 cm i.d.). Under sterile conditions and constant anoxic gas flow (100% N2) 1.75 cm3 anoxic Hoagland solution (100%, 35 °C, pH 6.8) amended with 500 μM Fe(II)aq (from FeCl2) and 0.3% Gelrite (Carl Roth, Karlsruhe, Germany) were filled into the containers. When cooling down to room temperature, the solution formed a transparent solid soil matrix. Approximately 100 mL of 20% Hoagland solution was constantly kept on top of the growth gel to prevent desiccation. The growth containers were wrapped in aluminum foil to protect the soil matrix from illumination. The leaf biomass, however, was illuminated by light. These setups were kept in a specifically-designed greenhouse to maintain constant light, temperature and humidity conditions. Penetration of O2 from the atmosphere into the gel was monitored by microelectrodes and found to be relevant only in the upper 0.7 cm of the growth gel and therefore considered to be neglectable for the investigation of the rhizosphere.”
Line 85: Perhaps rephrase to sterile 50% Hoagland solution, as it is phrased this way on line 92.
We fully agree with the reviewer’s suggestion and made changes to the corresponding sentence in the manuscript.
[L.88] “…seedlings were pre-grown in sterile 50% Hoagland solution at pH 6.8”
Lines 112-119: Is it necessary to define both M and Airon plaque if they are the same thing? Perhaps better to go with convention (don’t think there is one) or only use one variable. I would also move the equation for Aimage to its own line following its introduction on line 110.
We agree that the derivation of equations was unnecessarily complicated and might by misinterpreted by readers following two variables with the same meaning. In order to precisely annotate the image area & derived iron plaque are, we modified equations (1)-(3) in the manuscript as follows:
[L.112-122]: The surface of root iron plaque minerals was estimated by extrapolating the 2-dimensional area of iron plaque formation (Aimage) following:
|
Aimage = 2 * r * h = d * h |
(1) |
with h (cm) as imaging area height, r (cm) as imaging area radius and d (cm) as imaging area diameter to a 3-dimensional outer surface mantle area of ideal cylindrical roots [37] as an approximation for the mineral surface of root iron plaque (Airon plaque) following:
|
Airon plaque = 2 * r * ? * h |
(2) |
summarized in the following simplified equation as:
|
Airon plaque = Aimage * ? |
(3) |
Line 132: Should PFA be spelled out?
We appreciate the reviewer’s comment and agree that the abbreviation for paraformaldehyde was not introduced before. We therefore modified the manuscript accordingly:
[L.136]: “In order to inhibit cell activity in abiotic control incubations, 4% paraformaldehyde (PFA) were added to the respective setups.”
Lines 133 & 140: How long were these incubations?
We fully agree that the duration was missing for the Fe(III) reduction incubation and added the incubation time of 8 days into the corresponding section in the manuscript.
[L.137-138]: “Microcosms were kept in the dark at constant temperature (24°C) for a total of 8 days.”
Line 135: This sentence is confusing, presumably roots covered in plaque were sampled from a 52 day old plant, but the way it reads it sounds like you’re dunking them in root plaque.
Yes, indeed. We agree that the way this sentence reads can be misleading. In order to eliminate a misinterpretation and to clarify the experimental approach we split the long sentence into two individual parts and removed displaced words in the manuscript.
[L.140-143]: “Roots covered in iron plaque were sampled from a 52-day old rice plant which was previously grown on an Fe(II)-rich hydroponic solution. Collected root material was soaked in a cell suspension of an Fe(III)-reducing enrichment culture (see above) and consequently transferred into a rhizotron that contained warm (35°C), sterile and anoxic mineral medium [38], amended with 0.3% Gelrite.”
Line 159: DAT is introduced here, but not defined until page 5.
Line 159: I don’t know that whiff is the best word, maybe “intermittent oxygen was detected”
We are thankful for this comment and suggestion and agree that the abbreviation was introduced too late in the manuscript. Additionally, we think the suggested termination for “whiffs” is definitely more appropriate in the respective sentence. We added the explanation for DAT and modified the sentence accordingly:
[L.165-167]: “After 5 days after transfer (DAT), intermittent O2 was detected in the rhizosphere forming steep gradients ranging from 70 µM O2 on the surface of first lateral root tips to <5 µM O2 expanding 6 mm into the anoxic soil matrix.”
Line 198 / Figure 3: As you astutely pointed out, both the average total root surface area and the surface area of root iron plaque increased, but I think it would be more informative to normalize the amount of root iron plaque increase by the amount of total surface area available at the different measuring points and could warrant moving the supplemental figure (S1B) to the paper itself. Figure 3A is somewhat misleading at a cursory glance since it makes it seem that the overall root coverage is increasing over time, while S1B shows that the average root covering is proportionally more consistent.
We highly appreciate the reviewer’s suggestion and agree that the relative root area covered in iron plaque VS non-covered root surface deserves more attention. We therefore moved Figure S1 B from the Supporting Information into the main text replacing Figures 3B-G.
Moreover, we realized that Figures 3B-G were not considerably mentioned in the main text and therefore moved Figures 3B-G from the main text into the Supporting Information.
Additionally, we modified the respective figure captions and added information about relative root iron plaque coverage into the caption of Figure 3.
In the main text, information on the observation requested by the reviewer now appears in L.208-213:
[L.208-213]: “However, among all replicate setups, the ratio of iron plaque covered to non-covered root surface area widely varied within 30 DAT ranging from <15% to more than 50% of the total root surface covered in iron plaque precipitates (Figure 3B). Yet, towards the end of the experiment after 45 DAT, on average 36 (±2.6) % of the total root surface area in all setups were covered in iron plaque minerals which corresponds to an average root iron plaque surface area of 75 (±13.6) cm2 per plant at the end of the experiment.”
[L.516-517]: “Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Root surface, iron plaque area and evolution of root iron plaque during rice plant growth;”
|
Figure 3 – Root iron plaque formation during plant growth. A: Iron plaque mineral surface (cm2 per plant) on roots during plant growth (DAT = days after transfer) of five replicate plants (filled symbols), mean root iron plaque surface area (cross & punctuated line) and standard deviation (grey bars); B-D: Root growth and iron plaque formation in rhizotrons 5, 18 and 30 DAT; B: Ratios of iron plaque-covered to non-covered root surface area (%) in rhizotrons during plant growth over 45 DAT, averaged ratio of all replicates (cross & punctuated line) and standard deviation (grey bars). |
|
Figure 5: At which incubation time point was the photo for panel A taken? (e.g. after 43 hours incubation)
We fully agree that the information in incubation duration was missing in the figure and added the respective time of 24 hours into the figure caption of Figure 5, that now states:
[Figure 5] “A: Root iron plaque exposed for 24 hours to an Fe(III)-reducing enrichment culture (99.8% similarity to Geobacter spp.). Iron plaque minerals changed from orange (control) to black (biotic) during incubation.”
Figure 6: Include in figure legend for 6F which timepoint measurements are for (day 10).
We agree that this information was missing and added the time point of incubation to the respective figure legend of Figure 6 that now states:
[Figure 6] “F: Voltammetric measurements along transect a-b in setup of Figure, after 10 days of incubation, detect Fe(II) remobilized from root iron plaque is closely associated with roots.”
Line 287: Improper use of the word “amount,” try “the large number of iron minerals…”
We appreciate the language suggestion and modified the main text accordingly.
[L.294-296]: “Evidently, this large number of iron minerals that can precipitate on the root surface has broad consequences not only for the immobility of soil-borne iron but the biogeochemical cycling of iron in paddy soils.”
Figure 7: Similar concerns with the inset graph that is from Figure 3. Also, despite having talked about them earlier you are missing, for example, microaerophilic FeOB in your summary of the rhizosphere iron cycle. If you want to only show data from this study, consider rephrasing the figure title. This could also address my other concern, that the figure implies that only Geobacter spp. are involved in iron reduction in the plant rhizosphere. You may also consider moving Figure 7 to before the introduction of microaerophilic FeOB in the discussion altogether, as that would make the omission less glaringly obvious.
We appreciate the reviewer’s suggestion to reconsider changing the title of Figure 7, in order to clarify that Figure 7 is only displaying biogeochemical processes observed in the current study. Moreover we agree that the impact of microaerophilic Fe(II)-oxidizing bacteria is not shown in this figure. However, as the title now suggests, only observed and therefore proposed processes are shown in Figure 7 which does not include microaerophilic Fe(II) oxidation and the capability to adsorb other soil constituents such as arsenic.
We therefore changed the title of Figure 7 to clarify that this figure is only summarizing processes observed in the current study and made additional changes to stress this out in the respective figure caption that. Additionally, we moved Figure 7 before the discussion on microbial impacts on the paddy field iron cycle. Figure 7 caption now reads:
[Figure 7] “Figure 7 – Summary of biogeochemical processes observed in this study that affect the rhizosphere iron cycle at and around rice roots covered in root iron plaque. Left: Oxidative side of the outlined rhizosphere iron cycle with estimates for an immobilization of iron per gram dry weight rice root, root iron plaque coverage and diurnal changes in ROL at root tips that affect local redox conditions. Right: Reductive side of the proposed rhizosphere iron cycle with observed rates and extent for root iron plaque remobilization through reductive dissolution and reductive mineral transformation by the Geobacter spp. culture used in the current study, affecting iron plaque crystallinity and bioavailability, respectively.”
Line 479/480: “… would find their ideal niches conditions, even enhance Fe(II) oxidation…” needs rephrasing. Not entirely sure what the connotation of the statement was supposed to be.
We agree that this sentence was probably over-corrected too often, now losing its statement. We therefore re-consolidated the message by rephrasing the respective sentence in the manuscript.
[L.504-507]: “Microaerophilic Fe(II)-oxidizing bacteria, however, can find their ideal niche conditions in the opposing gradients of soil-borne Fe(II) and O2 from ROL [15, 17] and might even enhance Fe(II) oxidation kinetics [33] and consequently contribute to root iron plaque formation [18].”
Lines 310-338: The arsenic discussion is a bit over-reaching as no heavy metal studies were done here. Please streamline section and limit it to general implications that your data can make.
Here, we only partly agree with the reviewer’s suggestion. Numerous studies demonstrated that iron plaque can immobilize metal(oid)s, such as arsenic, by complexation or adsorption. However, to date an enumerative understanding on how much initially dissolved arsenic can be immobilized on root iron plaque is still lacking. The aim of this discussion was to provide evidence that the capability of rice plants to produce ferric root iron plaque minerals can significantly impact the total arsenic budget in contaminated paddy fields. This simulated budgeting of surface sites and binding capacities should highlight the role of iron plaque precipitates in the contaminant cycling in rice paddies. In particular with regards to reviewer’s first suggestion to “integrate a more wholistic view”, we consider the arsenic problem (omnipresent in numerous paddy fields) as absolute necessity to be discussed in this context.
Therefore, we are tempted to leave this section in the discussion part about the role of rice roots as initiator for rhizosphere iron mineral formation and heavy metal immobilization. We hope that the quantitative approach to estimate arsenic immobilization capacities of root iron plaque stimulates future research to develop quantitative approaches to not only follow the arsenic translocation from field to grain but rather consider paddy field biogeochemical processes that affect the entire paddy soil horizon as an entire ecosystem.
However, we are open for discussion with the respective reviewer and are convinced we will find an appropriate compromise to address this message.
However, we agree that we clearly need to point out that the discussion is based solely on the calculated assumption and not on observed arsenic data. Therefore, we modified the main text and clearly stated that the observed iron plaque formation was considered to estimate potential effects on the retention of arsenic in contaminated rice paddies.
[L.316-328] “…The formation of plant-induced iron (oxyhydr)oxide minerals in the soil horizon could theoretically impact also the retention of other soil constituents and metal(loid)s such as arsenic. In other words, under the theoretic assumption that one dissolved soil-borne ion can bind to one surface group of root plaque ferrihydrite, the observed formation of root iron plaque on the roots of rice plants could provide surface sites for approx. 0.2 µmoles dissolved ions per day within one m3 of paddy soil. Typically, paddy fields contaminated with arsenic contain up to 150 µg arsenic (=2 µM) per liter pore water. Given an effective soil porosity of 0.25, which is commonly observed in wetted paddy fields [47], the pore water of 1 m3 paddy soil can effectively contain 500 µmoles of arsenic ions. Based on these assumptions and the observed root iron plaque formation from the current study, the roots of 100 rice plants on one 1 m2 paddy field would have the potential to form iron mineral surface sites that can bind and immobilize up to 20 µmoles of arsenic per growing season, which corresponds to approx. 5% of the total dissolved arsenic in 1 m3 contaminated paddy soil.”
Line 487-488: Please re-phrase as your research did not specifically show this, but the sentence implies that it is based on the results of this study itself.
Yes, we are thankful for the reviewer’s comment and fully agree that this sentence misleads the reader to wrong conclusions drawn from this manuscript. In order to emphasize that this study does not show these results but rather concludes to processes that can happen in the environment, we modified the respective section in the main text.
[L.514-516]: “In the paddy soil environment, the total root iron mineral surface area which increased with plant age (Figure 2) can consequently affect the retention of nutrients and has the potential to immobilize heavy metals
Reviewer 2 Report
This manuscript described a creative study that not only showed the iron plaque formation process on a very fine scale, but also intended to apply the calculated parameters to simulate iron cycle budget in the paddy rice system. Generally the manuscript was very well written with some novel imaging techniques involved. The reviewer, however, would like to point out the potential biases using laboratory-generated data to calculate/predict field-scale iron or other elements cycle budget. First, the authors chose only one Fe(II) initial concentration (500 uM), which cannot differentiate high Fe and low Fe soil (clays). Second, use of model compounds in the aqueous solution, e.g., Na-acetate (which is a low molecular weight and relatively labile organic chemical) as the electron donor, instead of naturally existing organics in the soil (such as large molecule sugar/protein/fat or recalcitrant humic substances), might have over-simplified the biochemistry in the paddy system. Finally, the study only used one rice variety without considering the impacts of irrigation practices (e.g. continuous flooding vs. alternative wetting and drying). These factors would all influence the yield, composition and therefore (physio)chemical properties of the iron plaque. Consequently, cautions are needed when using lab-based data to simulate field situations. It would be great if an extra paragraph could be added emphasizing these issues or proposing to set up more complex simulations to mimic in-situ field conditions as future research direction.
Author Response
Comments and suggestions for Authors from Reviewer 1
This manuscript described a creative study that not only showed the iron plaque formation process on a very fine scale, but also intended to apply the calculated parameters to simulate iron cycle budget in the paddy rice system. Generally the manuscript was very well written with some novel imaging techniques involved. The reviewer, however, would like to point out the potential biases using laboratory-generated data to calculate/predict field-scale iron or other elements cycle budget. First, the authors chose only one Fe(II) initial concentration (500 uM), which cannot differentiate high Fe and low Fe soil (clays).
We appreciate the reviewer’s comment and agree that the experimental outline with only one concentration of Fe(II) as treatment might diminish possibility to extrapolate the observations to all paddy soils in general. We consequently also agree that the availability of Fe(II) ultimately affects iron plaque formation and the impact on other soil parameters. However, in order to systematically investigate the formation of root iron plaque, we deliberately chose only one Fe(II) concentration for all incubations.
However, in order to address the reviewer’s excellent suggestion, we added a statement to the discussion on Rice roots as initiator for rhizosphere iron mineral formation and heavy metal immobilization and pointed out that Fe(II) concentrations in rice paddies can differ substantially.
[L.327-335]: “…A large variety of dissolved and mobile soil components, such as phosphate, magnesium, soil organic matter and other chelating compounds [49] might compete for surface binding sites on the root iron plaque and potentially decrease the net amount of arsenic binding to root iron plaque. Moreover, as ferrous iron concentrations in rice paddies can vary to a large extent, the reported estimates for the formation of root iron plaque minerals potentially only apply for soil parameters with similar, rather elevated, iron concentrations. In paddy soils with a relatively high abundance of clay minerals, dissolved Fe(II) concentrations were reported to be lower, thus diminishing the formation of root iron plaque minerals.”
Second, use of model compounds in the aqueous solution, e.g., Na-acetate (which is a low molecular weight and relatively labile organic chemical) as the electron donor, instead of naturally existing organics in the soil (such as large molecule sugar/protein/fat or recalcitrant humic substances), might have over-simplified the biochemistry in the paddy system.
We fully agree with the reviewer’s valuable suggestion and agree that sodium acetate represents only a small fraction of soil organic matter available to Fe(III)-reducing bacteria. The Fe(III)-reducing culture (99.6% similar Geobacter spp.) isolated from a paddy soil exclusively uses acetate as electron donor substrate to reduce Fe(III). We therefore amended the Fe(III)-reducing incubation with acetate to simulate the activity of one representative among the Fe(III)-reducing community of bacteria in our simplified soil matrix.
However, we agree that future research should definitely increase the complexity and integrity of this rhizosphere analogue towards environmental conditions, using more complex metabolic substrates such as extracted NOM, SOM or humic substances for the Fe(III)-reducing community.
We therefore included the reviewer’s suggestion into the discussion on the environmental relevance and stress out, that future studies should potentially use the same experimental approach but increase the complexity towards more environmental conditions.
[L.534-540]: “…We suggest future research to include similar approaches, increasing the integrity of biogeochemical interactions to fully decipher potential cross-links in the iron cycle between the enormous variety of participants. One potential step towards an understanding of the iron cycle in a more complex rhizosphere system could be the quantitative spatiotemporal investigation of root iron plaque formation incubated in the presence and absence of different members of iron-cycling bacteria, and the availability of soil-extracted soil organic matter or humic substances as metabolic substrate.”
Finally, the study only used one rice variety without considering the impacts of irrigation practices (e.g. continuous flooding vs. alternative wetting and drying). These factors would all influence the yield, composition and therefore (physio)chemical properties of the iron plaque. Consequently, cautions are needed when using lab-based data to simulate field situations. It would be great if an extra paragraph could be added emphasizing these issues or proposing to set up more complex simulations to mimic in-situ field conditions as future research direction.
We are thankful for the reviewer’s argument and fully agree that a more wholistic view on variable parameters was lacking in the current form of the manuscript. We modified the main text and included a statement where we emphasize the simulation of different irrigation strategies to future research motivating the reader to increase the complexity of the current study to increase the understanding of the complex rhizosphere iron cycle in rice paddies.
[L.540-547]: “…Moreover, also different irrigation practices could be simulated in future studies. Soil redox conditions in the current setup were maintained constantly anoxic, representing water-logged paddy field. Periodic draining of paddy fields, however, might switch redox-conditions to prevalent oxic conditions, suppressing microbial Fe(III) reduction. Taken all these variable parameters together, the understanding of this spatio-dynamic small-scale iron redox rhizosphere system can have a huge impact on large scale observations and should be considered for future investigations of the rhizosphere iron redox cycle in paddy fields.”
and contaminants to a relevant extent.”
Round 2
Reviewer 1 Report
The authors have thoroughly addressed the reviewer's comments updating the figures, expanded the discussion, and clarifying necessary information. These changes have improved the manuscript and are sufficient for publication in Soil Systems.
Author Response
We want to thank the reviewer for substantially improving the quality of our manuscript.
