Photosynthetic Acclimation and Growth Responses to Elevated CO₂ Associate with Leaf Nitrogen and Phosphorus Concentrations in Mulberry (Morus multicaulis Perr.)

Round 1

Reviewer 1 Report

The manuscript is focused on the effect of elevated CO₂ on growth, physiology and nutrient uptake of two mulberry varieties. The presented manuscript contains the results of a pot experiment, the results are clear, conclusive and well described. The discussion is also sufficient and I have basically no problem with the manuscript in the present form.

I have only some minor comments and recommendations for the authors:

Introduction:

This caption is clear and well written.

 

Methods:

The methodology is simple and straight forward and therefore effective.

P3 L101-103: You should write the term of planting. We don´t know how long the plants were exposed to different amount of CO₂ in the growth chambers.

P3 L118-125: You should explain at what CO₂ concentration was the physiological parameters measured? ACO₂ at 410 ppm and eCO₂ at 710 ppm?

P3 L118: Why do you mention that you measured the photosynthesis on sunny days when the experiment took place in growth chambers?

 

Results:

This caption is clear and well written. The figures clearly show the results achieved.

 

Discussion:

P9 L290-305: You should also take into account the effects of the measurement term on physiological measurements and condition of plant samples (end of the experiment). Probably the photosynthesis in eCO2 was higher during vegetative plant growth in comparison with ACO₂ and only after the depletion of nitrogen in this variant you found reduction in photosynthesis.

Author Response

The manuscript is focused on the effect of elevated CO₂ on growth, physiology and nutrient uptake of two mulberry varieties. The presented manuscript contains the results of a pot experiment, the results are clear, conclusive and well described. The discussion is also sufficient and I have basically no problem with the manuscript in the present form.

I have only some minor comments and recommendations for the authors:

Introduction:

This caption is clear and well written.

Response: Thank you for your encouragement.

 

Methods:

The methodology is simple and straight forward and therefore effective.

P3 L101-103: You should write the term of planting. We don´t know how long the plants were exposed to different amount of CO₂ in the growth chambers.

Response: Thank you for your comments. The sentence of Line 101-103 “Two seedlings per variety were planted in a plastic pot (20 x 32 cm = height x diameter) filled with 11 kg soil (Eutric Regosol, FAO Soil Classification System) on May 10, 2020.” has been changed to “Two seedlings per variety were planted in a plastic pot (20 x 32 cm = height x diameter) filled with 11 kg soil (Eutric Regosol, FAO Soil Classification System) from May 10 to September 16, 2020.” Moreover, the sentence of Line 111 “The plants were harvested on September 16, 2020” has been changed to “The plants were harvested on September 16, 2020 after 129 days of CO₂ exposure.”

 

 

P3 L118-125: You should explain at what CO₂ concentration was the physiological parameters measured? ACO₂ at 410 ppm and eCO₂ at 710 ppm?

Response: Thanks for your concern. We have added the following writings of “Leaf gas exchange parameters were measured at 410 ppm and 710 ppm CO₂ for plants grown under ACO₂ and eCO₂, respectively” in the line of 138-139 in the R1 reversion.

 

 

P3 L118: Why do you mention that you measured the photosynthesis on sunny days when the experiment took place in growth chambers?

Response: Thanks for your comment. The growth chambers were constructed by tempered glasses (10 mm thickness, 90 % light transmission rate) and installed in open fields, not a light-manipulated green-house, at the National Monitoring Station of Soil Fertility and Fertilizer Efficiency on Purple Soils. The detailed information has been now added in the Experiment Design and the Supplementary material (Figure S1A-B).

 

Results:

This caption is clear and well written. The figures clearly show the results achieved.

Response: Thanks for your encouragement.

 

Discussion:

P9 L290-305: You should also take into account the effects of the measurement term on physiological measurements and condition of plant samples (end of the experiment). Probably the photosynthesis in eCO2 was higher during vegetative plant growth in comparison with ACO₂ and only after the depletion of nitrogen in this variant you found reduction in photosynthesis.

Response: Thank you for your valuable comment. The previous discussion “In addition, the effect of eCO₂ on plant biomass production has been often explored in combination with photosynthesis, which caused accumulation of assimilates in plant organs [43]. However, our results showed that eCO₂ caused a down-regulation or decline of photosynthetic capacity and hence evidenced photosynthetic acclimation (Figure 1A). Similar observations have been made in Populus deltoides seedlings grown under 700 ppm eCO₂ in a pot experiment [20]. The positive relationships between net photosynthetic rate and leaf N or P concentration under eCO₂ (Figure 6D - 6E) suggested that a photosynthetic down-regulation was due to reductions of resource availability. We observed that 18 %-22 % and 7 %-10 % of leaf N and P concentrations were decreased, and N and P partitioning into stems were increased in both mulberry varieties under eCO₂ (Figure 5A - 5B). Since the RuBisco amount in a leaf is determined by N allocation to the leaf [8], the fraction of N allocated to RuBisco was likely decreased under eCO₂ [20, 44]. Although chlorophyll a+b concentration was decreased by eCO₂ (Figure 2C), it was significantly positively related to leaf N concentration (Figure 6A), indicated that under 710/760 ppm eCO₂ N limitation was the cause of photosynthetic acclimation and the degree of photosynthetic acclimation was more pronounced in N-deficient plants [45].” has been change to “The effect of eCO₂ on plant biomass production largely occurs through increased photosynthetic rates [47], and the mechanisms of photosynthetic stimulation by eCO₂ have been reported in many species, such as Artemisia annua [41], Camellia sinensis [48], cassava [49], grapevine [2] and Tabebuia rosea [50]. In mulberry, leaf photosynthetic rates were greatly increased by 27 %-32 % by 550-800 ppm eCO₂ [31-33]. This is because under ACO₂, CO₂ supply is often a limit to growth and eCO₂ accelerates carboxylation processes. However, a down-regulation of leaf photosynthesis was also observed in pot experiments [20, 51] or under field conditions [3]. The effect of eCO₂ on growth and photosynthesis was variable throughout plant growth stage and/or time of CO₂ exposure. For example, compared to Glycine max grown under ACO₂, photosynthetic rates were higher after 8 weeks, while much lower after 12 weeks of exposure to 1,000 ppm eCO₂ [52]. Carbon gain in rice under 695 ppm eCO₂ was increased by 22–79 % during the vegetative growth, but decreased to -12–+5 % after grain-filling, leading to a 7–22 % net increase for the whole season [53]. Net photosynthetic rate of Lycium barbarum displayed a downward trend at 90 and 120 days under 760 ppm eCO₂ [54]. The positive effects of 600 ppm eCO₂ on growth and chlorophyll content were greater in 20 days old than in 40 days old plants, but not on those in 41-65 days old mungbean plants [55]. Similarly, a down-regulation or decline of photosynthetic capacity and chlorophyll concentration for such photosynthetic acclimations were also evidenced for ~120 days old mulberry plants under 710 ppm eCO₂ (Figure 1A and Figure 2). The following mechanisms could explain these decreases in photosynthetic parameters under eCO₂, as the limited pot space had most likely restricted both the growth and functioning of the root system, leading to a decrease of nutrient uptake and hence a decreased movement of photosynthates to roots. Soil nutrients might also not be sufficient for matching up with CO₂ assimilation or photosynthesis to greater plant biomass production under eCO₂. We observed that 18 %–22 % and 7 %–10 % of leaf N and P concentrations were decreased, and N and P partitioning into stems were increased in both mulberry varieties under eCO₂ (Figure 5A-5B). Greater positive relationships between net photosynthetic rate and leaf N concentration under ACO₂ than under eCO₂ (Figure 6D) confirmed that a photosynthetic down-regulation was due to reductions of resource availability [56]. Since RuBisco protein is determined by leaf N allocation [8], the fraction of N allocated to RuBisco would be decreased under eCO₂ [20, 53], leading to deficiencies in both amount and activity of Rubisco protein or surplus of C for synthesis of secondary compounds under eCO₂ [57-58]. Moreover, significantly positively greater relationships between leaf N concentration and chlorophyll a+b, net photosynthesis rate or transpiration rate under 410/460 ppm CO₂ than under 710/760 ppm eCO₂ (Figure 6A, 6D and 6G), indicating that N limitation under eCO₂ was the cause of photosynthetic acclimation, which was more pronounced in N-deficient plants [59]. Furthermore, eCO₂ and low N supply decreased activities of some antioxidant enzymes and thus increased accumulation of reactive oxygen species [60-61]. These changes in oxidative stress could accelerate the degradation of chlorophyll (Figure 3) and eventually induce senescence [62], while eCO₂‐induced changes were mainly displayed as a general down‐regulation of leaf carbohydrate metabolism [57-58]. Nevertheless, net assimilation rates measured at 350 or at 700 ppm CO₂ were not significantly different, neither [59]” in the line of 330-373 in the R1 reversion.

Author Response File: Author Response.doc

Reviewer 2 Report

Songmei Shi  Forests Photosynthetic Acclimation and Growth Responses to Elevated  CO2 Associate with Leaf Nitrogen and Phosphorus Concentrations in Mulberry (Morus multicaulis Perr.)

I am puzzled by this paper.   Nearly all nutritional variables increased under eCO2 yet the photosynthetic rates and stomatal conductance were reported to be reduced.   The authors report a very large increase in growth and tissues sizes in the plants exposed to elevated CO2 (Table 1).   The increase in growth should be the largest message from this work but it appears that it is hardly worth mentioning in the Discussion.   Indeed, I cannot image how such growth was achieved if there was such a large decline in net Ps.    Does the Ps measured at the end of the experiment reflect conditions during the growth phase?   Or was the photosynthesis of eCO2 trees measured at the lower aCO2 conditions in the cuvette?   It is not clear in the Methods section.   Regardless, the authors seem to content to track the previous literature instead of highlighting the large increases in growth seen in this study.  Instead, there should be effort to explain this positive growth.    As growth is an easy variable to measure (and is likely has the fewest errors in measurement), it must be taken very seriously.

 

L92 were placed in each chamber

L168 Was the CO2 level in the cuvette held at the appropriate treatment levels during measurements? This issue is critical to the value of the paper.

Fig. 5 reverse the order of the root, stem, leaf symbols caption to correspond with the graph.

Fig. 6  I am puzzled as to how Net Ps and transpiration are higher in the eCO2 here compared to the means presented in Fig. 1   Are the symbols reversed in Fig. 6?   Likely also applies to Fig. 7.

Author Response

I am puzzled by this paper.   Nearly all nutritional variables increased under eCO2 yet the photosynthetic rates and stomatal conductance were reported to be reduced.   The authors report a very large increase in growth and tissues sizes in the plants exposed to elevated CO2 (Table 1).   The increase in growth should be the largest message from this work but it appears that it is hardly worth mentioning in the Discussion.   Indeed, I cannot image how such growth was achieved if there was such a large decline in net Ps.    Does the Ps measured at the end of the experiment reflect conditions during the growth phase?   Or was the photosynthesis of eCO2 trees measured at the lower aCO2 conditions in the cuvette?   It is not clear in the Methods section.   Regardless, the authors seem to content to track the previous literature instead of highlighting the large increases in growth seen in this study.  Instead, there should be effort to explain this positive growth.    As growth is an easy variable to measure (and is likely has the fewest errors in measurement), it must be taken very seriously.

Response: Thanks for your valuable comments.

 

1. With respected to enhanced plant growth or biomass production under eCO₂, please see our discussions in the first paragraphs in the Discussion section. We have also added the following writings of “Numerous studies on a variety of plant species have demonstrated that biomass production was generally enhanced under a range of eCO₂ than under ACO₂ [34-35, 43-44].” in Line 297-299 of the R1 version, which we cannot discuss their details in the main text due to space limitation.

 

2. The previous sentence of “The measurements were taken during the period of 9:00-11:00 a.m. on sunny days before harvest.” has been changed to “The measurements were taken before harvest during the period of 9:00-11:00 a.m. on sunny days of September 8, 11 and 15, 2020 (Data were averaged from these three days)” (See Line 132-134 in the R1 version).

 

3. We have added the following writings of “Leaf gas exchange parameters were measured at 410 ppm and 710 ppm CO₂ for plants grown under ACO₂ and eCO₂, respectively” in the line of 138-139 in the R1 reversion.

 

 

L92 were placed in each chamber

Response: Corrected as suggested, thanks.

 

L168 Was the CO2 level in the cuvette held at the appropriate treatment levels during measurements? This issue is critical to the value of the paper.

Response: Thanks for your concern. We have added the following writings of “Leaf gas exchange parameters were measured at 410 ppm and 710 ppm CO₂ for plants grown under ACO₂ and eCO₂, respectively” in the line of 138-13 in the R1 reversion.

 

Fig. 5 reverse the order of the root, stem, leaf symbols caption to correspond with the graph.

Response: Done as suggested, thanks.

 

Fig. 6 I am puzzled as to how Net Ps and transpiration are higher in the eCO2 here compared to the means presented in Fig. 1   Are the symbols reversed in Fig. 6?   Likely also applies to Fig. 7.

Response: Thanks for your comment. Sorry for the mistake. The symbols of Fig.6 and Fig.7 have been reversed in the R1 version.

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

I had a look at the revised version.  It addresses my largest concerns with the paper.