Growth and Physiological Responses of Norway Spruce (Picea abies (L.) H. Karst) Supplemented with Monochromatic Red, Blue and Far-Red Light

Round 1

Reviewer 1 Report

The current ms describes the observed effects of different light spectra on the growth and physiologial factors in three-year old Norway spruce plants. The ms contains a lot of information, but interpretation of the data is difficult due to couple of fundamental issues not explained well enough.

My major concerns are related to the material used, as well as experimental set up:

• The spruce plants are said to be "clones" of Czech origin. How were they produced, i.e. were they rooted cuttings, emblings or grafts ? Were they all of a single genotype, or how many genotypes were involved ? if there were several genotypes, how many, and how these genotypes were divided among the treatments? Were any genotypic effects seen ?  if they were several genotypes used, you need to include also genotypic effects on analyses...
• What was the original latitude of the clonal material, and how close it was to the latitude of the research place ? The authors discuss the effect of latitude but do not tell their own case... 
• Why did you not have 0-control i.e. plants grown without any additional light ??
• Did you measure the plants (height, diameter)  at the starting time of the experiments ? Without this measurement you have no data showing that the plant material in different treatments were similar, and you cannot compare the treatments...

Another concern is the way in which the results are often presented: I.e. differences among the treatments are told in the text - but without stating that they are not significant (as seen in the figures). This may be misleading. There are also several points in which text and the figure are not consistent  (e.g. r 282-283). Please also try to avoid repeating all the results in the text when showing them in figures.

The ms contains unfortunately also many points that have been written in careless manner:

• In gas-exchange measurements the authors claim to measure six plants per species.. were there other species than Norway spruce ?
• The light treatments appear in varying order in different tables and figures making it very difficult fo the reader to follow them. Please use the same order consistantly in all the figures and tables.
• Table 2 is unclear. Revise so that the all studied traits are clearly shown with their own variance sources and not mixed under one indicated title row… 
• check also references, e.g. 40 and 46 are the same

Author Response

The current ms describes the observed effects of different light spectra on the growth and physiological factors in three-year old Norway spruce plants. The ms contains a lot of information, but interpretation of the data is difficult due to couple of fundamental issues not explained well enough.

My major concerns are related to the material used, as well as experimental set up:

• The spruce plants are said to be "clones" of Czech origin. How were they produced, i.e. were they rooted cuttings, emblings or grafts ? Were they all of a single genotype, or how many genotypes were involved ? if there were several genotypes, how many, and how these genotypes were divided among the treatments? Were any genotypic effects seen?  if they were several genotypes used, you need to include also genotypic effects on analyses...

Response: Thank you for your good suggestion. They were rooted cuttings and all of them were from a single genotype. It should be cutting orchard of Norway spruce was established from the seedlings of Czech origin. The shoot cuttings were harvested from a single clone of the cutting orchard.

• What was the original latitude of the clonal material, and how close it was to the latitude of the research place ? The authors discuss the effect of latitude but do not tell their own case... 

Response: Thank you for your good suggestion. The original location of the clonal material was E 14°25′15″, N 50°13′13″ in the Czech Republic. The experiment site located in the Research Institute of Forestry of Xiaolongshan, Gansu Province (E 105°54′37″, N 34°28′50″) of China.

• Why did you not have 0-control i.e. plants grown without any additional light?

Response: Thank you for your good suggestion. The purpose of this study is to compare the effects of different light qualities on the growth and physiology response of Norway spruce cuttings. If Norway spruce grown without any additional light, it will stop growth with short growth period (50 d, Ouyang et al., 2020, Table 2). We conducted the complex light (the ratio of R:B:FR=7:1:1) as control. But, as your mentioned, a double control might be better.

Ouyang F , Ma J , Wang J , et al. Picea species from humid continental and temperate marine climates perform better in monsoonal areas of middle latitudes of China[J]. Journal of Forestry Research, 2020.

• Did you measure the plants (height, diameter) at the starting time of the experiments ? Without this measurement you have no data showing that the plant material in different treatments were similar, and you cannot compare the treatments...

Response: Thank you for your valuable suggestion. We measured the plants height and diameter at the starting time of the experiments. The data showed that there was no significant difference among the different treatments (P>0.05, Table 2). The results of multiple comparison (Duncan) were as follows:

Another concern is the way in which the results are often presented: I.e. differences among the treatments are told in the text - but without stating that they are not significant (as seen in the figures). This may be misleading. There are also several points in which text and the figure are not consistent  (e.g. r 282-283). Please also try to avoid repeating all the results in the text when showing them in figures.

Response: Thank you for your valuable suggestion. Line 282-283 should be ‘Nevertheless, significant differences were not observed in the expression of COP1-2, HY5, and PIF3 between the R treatments and the control. The expression value of COP1-2 were significantly lower compared with that of the FR light treatments.’.

We checked throughout the paper to avoid repeating and keep all the results consistence in the text and the figures.

The ms contains unfortunately also many points that have been written in careless manner:

• In gas-exchange measurements the authors claim to measure six plants per species.. were there other species than Norway spruce?

Response: Thank you for your valuable suggestion. It should be six rooting cuttings per clone. We revise it.

• The light treatments appear in varying order in different tables and figures making it very difficult for the reader to follow them. Please use the same order consistently in all the figures and tables.

Response: Thank you for your valuable suggestions. We checked throughout the paper to keep the order consistent in all the figures and tables.

• Table 2 is unclear. Revise so that the all studied traits are clearly shown with their own variance sources and not mixed under one indicated title row… 

Response: Thank you for your valuable suggestions. We revise Table 2 and Table 3.

Table 2. Summary of ANOVA results comparing Norway spruce morphological and physiological parameters among the four light spectrum treatments.

• check also references, e.g. 40 and 46 are the same

Response: Thank you for your good suggestion. We checked all of references and revised them. But 40 and 46 are different.  

40 Grassi, G.; Bagnaresi, U. Foliar morphological and physiological plasticity in Picea abies and Abies alba saplings along a natural light gradient. Tree Physiol. 2001, 21(12-13), 959-967.

46 Mccree, K.J. The action spectrum, absorptance and quantum yield of photosynthesis in crop plants. Agric. Meteorol. 1971, 9, 191-216.

Author Response File: Author Response.docx

Reviewer 2 Report

Review on forests-1039675

Light quality responses of woody species have been studied widely but there exist uncertainties on the effects of distinct wavelengths and the involvement of sensing foto-receptors and cross-talks of signal transduction pathways.

In the respect I found this study interesting and valuable, it aims to reveal the growth and physiological responses of an important forest species, Norway spruce, - at seedling age - under „semi-controlled” growth environment, using greenhouse facilities and supplemental illumination by red, far red and blue light. The concept of this study is not new: there were similar previous studies with different Norway spruce clones that evaluate the effects of origin of populations on the light quality responses. Knowing the results of former studies, the authors should describe a hypothesis why they conduct these experiments.

The title of manuscript is long and incomprehensible, should be shortened and improved There are lacking information in methodological section of the manuscript, the overall text needs language improvement, mistakes within the text and figures should be corrected.   

Abstract

line 27-29 the summary includes treatment information that is different from the method!

Methods

Several questions have arisen: You have to add information: Why did you selected this clone of Norway spruce for this study?  and why did use this origin?

You have to describe precisely the used light treatments and emphasize that these are supplemental treatments since the whole study was performed in a greenhouse.

It is also not clear what light quality was used as supplemental illumination – different wavelengths have been mentioned in the abstract and in the Methods section (line 103-105)

Concerning the phytochrome-mediated responses and the maximum absorbance wavelength of Pfr, 760 nm seems to be too high, it is more common to use 730nm. What was the reason of selecting 760 nm in this study?

You have to refer how red:far red ratio might influence the ratio of the active Pfr to the total P (Pfr+Pr) defined as the phytochrome photostationary state (PSS).

line 110 50 μmolm-2s -2 please correct to 50 μmolm-2s -1

Results

line 207-210 Fig 1 M : what does it sign (I presume that this is the control);  “the leaf area/leaf weight ratio (SLW, F)” – it is not correct, it is the SLA g dm-2 not SLW (LMA), but you also have to include the unit of this leaf trait.

line 205 You have also to check the trend of SLW (?)  and correct the sentence.

line 216 „GAs levels (Table 2), which were 13% higher with R (6.38 g/100ng) (Fig. 2): please correct to 6.38 ng/100g

line 227-232 this sentence is not clear- what is the chlorophyll content ratio and how does it differ from chlorophyll content?

line 251 Tr (7.04 µmol H2O2 m-2 s -1): you have to correct H2O and not H2O2!

line 307-308 You should include how the supplemental red light might influence the phytochrome photostationary state (PSS) in seedlings, using red light is presumably influence not only the photosynthesis but also growth traits

line 315-326 You mention the effect of origin on the light quality responses of Norway spruce. I suggest you have to compare your results to those of former studies obtained for Norway spruce populations of different latitudes.

line 346-348 difference in chlorophyll content among treatments also raises the question how the different treatments influenced the phytochrome photostationary state (PSS) in seedlings. Please consider this when you evaluate your results instead of mentioning not relevant papers like in line 353-355 Arabidopsis of different origin.

There are no real conclusions from your results. I wonder how these results can contribute to our knowledge on physiological performance of Norway spruce seedlings in plantations, in forest understory etc.

In summary, the manuscript, although it contains several valuable results, is very carelessly compiled, contains many errors, needs strong improvement in several respects. In the present form I cannot suggest to accept it for publication. I would also note that with this content I do not see a close connection of the manuscript to the profile of journal Forests, but rather to a physiological journal.

Author Response

Review 2

Review on forests-1039675

Light quality responses of woody species have been studied widely but there exist uncertainties on the effects of distinct wavelengths and the involvement of sensing photo-receptors and cross-talks of signal transduction pathways.

In the respect I found this study interesting and valuable, it aims to reveal the growth and physiological responses of an important forest species, Norway spruce, - at seedling age - under „semi-controlled” growth environment, using greenhouse facilities and supplemental illumination by red, far red and blue light. The concept of this study is not new: there were similar previous studies with different Norway spruce clones that evaluate the effects of origin of populations on the light quality responses. Knowing the results of former studies, the authors should describe a hypothesis why they conduct these experiments.

Response: I express my sincere gratitude for your valuable suggestion!  

A hypothesis why we conduct these experiments is: Although there were similar previous studies with different Norway spruce clones that evaluate the effects of origin of populations on the light quality responses [5,7-9,21], there exist uncertainties on the effects of distinct wavelengths on growth and physiological responses and the involvement of sensing the photo-receptors and cross-talks of signal transduction pathways.

The sentence “The study aims to reveal the growth and physiological responses of an important forest species, Norway spruce, - at seedling age- under “semi-controlled” growth environment, using greenhouse facilities and supplemental illumination by red, far red and blue light.” is added in our text to elucidate the aim of our study.

The title of manuscript is long and incomprehensible, should be shortened and improved. There are lacking information in methodological section of the manuscript, the overall text needs language improvement, mistakes within the text and figures should be corrected.   

Response: Thank you for your good suggestion. The title of manuscript revise “Growth and physiological responses of Norway spruce [Picea abies (L.) H. Karst] supplemented with monochromatic red, blue and far-red light”.

We add the details in the methodological section, polish the whole text and correct the mistakes within the text and figures according to your suggestion one by one.

Abstract

line 27-29 the summary includes treatment information that is different from the method!

Response: Thank you! We apologized for our carelessness. The summary has been revised:

“In the present study, three-year-old Norway spruce [Picea abies (L.) H. Karst] clones received sunlight and were illuminated for 12 h under LED light of monochrome blue light (B, 460 mm), monochrome red light (R, 660 nm), monochrome far-red light (FR, 730 nm) and complex light (control, the ratio of red light : blue light : far-red light is 7:1:1) light-emitting diode (LED) after sunset for 90 d, and their growth traits, physiological responses, and related gene expression were determined.”.

Methods

Several questions have arisen: You have to add information: Why did you selected this clone of Norway spruce for this study? and why did use this origin?

Response: Thank you. Previous experiments showed that the Czech provenance of Norway spruce had a good growth performance in the test site. The number of shoot cuttings of this clone were sufficient for evaluation. That’s why we selected this clone of Norway spruce for this study.

You have to describe precisely the used light treatments and emphasize that these are supplemental treatments since the whole study was performed in a greenhouse.

Response: Thank you. The clones normally received sunlight during the day and then illuminated after sunset for 12 h under LED light (Lian Bang Zhong Ke Electronic Technology Co., Ltd, Shenzhen, China). A timer was used to control the opening and closing of the lamps. The clones were subjected to light treatments with LED light in a greenhouse from May to August (90 d) in two continuous years. The four LED light sources were monochromatic red light (R, 660 nm), monochromatic blue light (B, 460 nm), monochromatic far-red light (FR, 730 nm) and complex light (control, the ratio of R:B:FR=7:1:1).

It is also not clear what light quality was used as supplemental illumination – different wavelengths have been mentioned in the abstract and in the Methods section (line 103-105)

Response: Thank you for your valuable suggestion. The summary was revised: “In the present study, three-year-old Norway spruce [Picea abies (L.) H. Karst] clones received sunlight during the day and were illuminated for 12 h under LED light of monochrome blue light (B, 460 mm), monochrome red light (R, 660 nm), monochrome far-red light (FR, 730 nm) and complex light (control, the ratio of red light : blue light : far-red light is 7:1:1) light-emitting diode (LED) after sunset for 90 d, and their growth traits, physiological responses, and related gene expression were determined.”.

The Methods section was revised: “ The four LED light sources were monochromatic blue light (B, 460 nm), monochromatic red light (R, 660 nm), monochromatic far-red light (FR, 730 nm) and complex light (the ratio of R:B:FR=7:1:1).”.

Concerning the phytochrome-mediated responses and the maximum absorbance wavelength of Pfr, 760 nm seems to be too high, it is more common to use 730nm. What was the reason of selecting 760 nm in this study?

Response: Thank you for your good suggestion. This is a mistake. It should be 730 nm.

You have to refer how red:far red ratio might influence the ratio of the active Pfr to the total P (Pfr+Pr) defined as the phytochrome photostationary state (PSS).

Response: Thank you for your valuable suggestions. The phytochrome photostationary state (PSS) is very important parameter. I add the following information about PSS in the Introduction section:

“Phytochromes exist in two forms: the red-light absorbing (Pr) form, which absorbs maximally at 660 nm and is generally considered to be biologically inactive; and the far-red light absorbing (Pfr), which absorbs maximally at 730 nm and is biologically active[4]. Absorption of light by either Pr or Pfr results in photo transformation between these two forms, which drives the on/off switching of the successive signaling pathway[5]. Phytochrome photostationary state (PSS) indicates the ratio of the active form of phytochrome (Pfr) to total phytochrome (Ptotal) (Pfr/Ptotal)[6]. As the FR ratio increases, i.e., R/FR decreases, PSS decreases due to the conversion of active Pfr to the inactive form, Pr. That is a low R/FR results in a low PSS [7-8].”

line 110 50 μmolm^-2s^-2 please correct to 50 μmolm^-2s ^-1

Response: Thank you. We correct it, it is 50 μmolm^-2s ^-1.

Results

line 207-210 Fig 1 M : what does it sign (I presume that this is the control);  “the leaf area/leaf weight ratio (SLW, F)” – it is not correct, it is the SLA g dm^-2 not SLW (LMA), but you also have to include the unit of this leaf trait.

Response: Thank you for your carefully checking. ‘Fig 1 M’ is the control. We change ‘control’ to ‘M’. We correct ‘SLW’ to ‘SLA’ and include the unit of this leaf trait (g dm^-2).

line 205 You have also to check the trend of SLW (?) and correct the sentence.

Response: Thank you. We replace the sentence by ‘The values of SLA in clones with R light treatment were significantly lower than that of the control, but they were no significant difference with those with B and FR light treatment..

line 216 „GAs levels (Table 2), which were 13% higher with R (6.38 g/100ng) (Fig. 2): please correct to 6.38 ng/100g

Response: Thank you for your carefully examination. We correct it to 6.38 ng/100g.

line 227-232 this sentence is not clear- what is the chlorophyll content ratio and how does it differ from chlorophyll content?

Response: Thank you. We correct ‘the ratio of leaf chlorophyll content to fresh weight’ to ‘the ratio of leaf chlorophyll to fresh weight’, that is the ratio of the leaf chlorophyll content to the leaf fresh weight, which is differ from chlorophyll content.

line 251 Tr (7.04 µmol H₂O₂ m^-2 s ^-1): you have to correct H₂O and not H₂O₂!

Response: Thank you for your carefully examination. We correct it to 7.04 µmol H₂O m^-2 s ^-1.

line 307-308 You should include how the supplemental red light might influence the phytochrome photostationary state (PSS) in seedlings, using red light is presumably influence not only the photosynthesis but also growth traits.

Response: Thank you for your valuable suggestions. The sentence “The previous studies showed PSS infuences plant physiology and morphology [47,7]. Using red light is presumably influence not only the photosynthesis but also growth traits, the supplemental red light might influence the PSS in Norway spruce cuttings. There is a positive correlation between the R/FR ratio and the PSS (Pfr/Ptot) levels [47,7-9]. According to this linear relationship, the PSS of the complex light with the ratio of R:B:FR=7:1:1 is roughly 0.82, while the PSS in R (0.85) and FR (0.03) in the present study. The maximum inhibition of Lactuca sativa seedlings hypocotyl growth at PSS ≈ 0.06 is due to the far-red high irradiance responses and depends on phyA. With the increasing or decreasing of PSS, the ratios of inhibition of hypocotyl growth were both decreased with different rate of decrease[47]. As such, we speculated maybe the PSS of R in our study was the best condition for Norway spruce growth among the four treatments.” was included in the ms.

line 315-326 You mention the effect of origin on the light quality responses of Norway spruce. I suggest you have to compare your results to those of former studies obtained for Norway spruce populations of different latitudes.

Response: Thank you for your valuable suggestions. We revised:

“the effect of red light and far-red light on Norway spruce growth seems to vary with latitude. Those from southern populations (59° N) are more sensitive to red light, even at low levels of radiation (0.1 Wm^-2) [8]. Studies have shown that growth requires far-red light for Norway spruce [6,7], but seedlings originating from 62°N latitude grew normally under the excluding far-red light treatment [8]. The latitude of the test site (34°N) was lower than the latitude of Czech provenance (50°N) where was the Norway spruce originated from. This suggested the Norway spruce may be more sensitive to red light. In conclusion, red light supplementation at night is effective for growth of Norway spruce clones.”

line 346-348 difference in chlorophyll content among treatments also raises the question how the different treatments influenced the phytochrome photostationary state (PSS) in seedlings. Please consider this when you evaluate your results instead of mentioning not relevant papers like in line 353-355 Arabidopsis of different origin.

Response: Thank you for your valuable suggestions. We delete line 353-355.

There are no real conclusions from your results. I wonder how these results can contribute to our knowledge on physiological performance of Norway spruce seedlings in plantations, in forest understory etc.

Response: Thank you for your valuable suggestions. The crown of spruce forest is dense and closed, thus the light under the canopy is weak, which is also called "dark coniferous forest". The ratio of red to far-red light (R/FR) below the plant canopy were greatly reduced, the light intenstiy also decreased. Under total light, the R: FR value was generally up to 1.02, and under the canopy, the R: FR value could be reduced to 0.2 [48]. So the PSS in a shaded place is lower than that above the plant canopy because most R light is absorbed by plants, and FR light is easily transmitted to below the plant canopy. Mølmann et al. (2006) showed One to-one mixtures of R and FR light were more effective in maintaining growth than either FR or R light alone, indicating a possible interaction between R and FR light maintaining growth[13]. Basing on the results, it may promote the regeneration of spruce forest under canopy, and accurately improve the forest quality by thining and pruning. In the future study, it may necessary to considerate the ratio of red to far-red light (R/FR) and determine the best PSS for Norway spruce seedlings growth.

In summary, the manuscript, although it contains several valuable results, is very carelessly compiled, contains many errors, needs strong improvement in several respects. In the present form I cannot suggest to accept it for publication. I would also note that with this content I do not see a close connection of the manuscript to the profile of journal Forests, but rather to a physiological journal.

Response: Thanks. We have modified carefully according to you and the other two reviewer’s suggestion. If there is anything that needs to be modified, please give us another chance to make modifications.  

Author Response File: Author Response.docx

Reviewer 3 Report

General impression

The manuscript comprises relatively high amount of data which are logically presented. However, the Authors do not indicate why they subjected the topic. Does the research has any practical implications, or is it just pure science?

The choose of R:B:FR 7:1:1 needs explanation. Was the 24-h or 12-h illumination implemented? Moreover, if 12-h photoperiod was implemented from May to August, such conditions were unnatural, hence the question arises why the experiment was performed such way?

The statement in the Abstract that the impact of the treatment needs further study is a little confusing. Materials and methods section should be improved as well as the figure captions.

The answers should be incorporated into the text of the future manuscript. At the moment it is not explanatory enough to be considered for publication.

Abstract

1.96%, 33.36% – such accuracy is unnecessary. Approx. 2%, 33% is quite enough and clearer.

Introduction

The first sentence sounds odd.

Materials and methods

Why the stock plants were of Czech origin?

Why R:B:FR was 7:1:1? Was the photoperiod 12-h or longer? From the sentence L106-107 it is unclear (24 hours?!).

The methods should be more explanative in most cases.

2.2. How leaf area was measured?

2.4. Please indicate more details.

2.5. Please indicate more details about protein measurement.

2.7. Please provide more details about the chlorophyll  fluorescence measurement protocol to allow the study to be reproducible (temperature, light-saturating pulse, actinic light, saturating pulses etc.).

Statistics: please indicate the probability level for the Duncan test.

Results

Figure captions: please indicate the meaning for B, R, FR, M.

Tables

Table 4: the values should be presented as 0.XXX to indicate all significant figures. At the moment, the results of the statistical test seems illogic in the case of : Fv/Fm.

Discussion

Based on the literature and the questions indicated in my general remarks, try to emphasise the novelty and innovativeness of your research.

Author Response

Review 3

General impression

The manuscript comprises relatively high amount of data which are logically presented. However, the Authors do not indicate why they subjected the topic. Does the research has any practical implications, or is it just pure science?

The choose of R:B:FR 7:1:1 needs explanation. Was the 24-h or 12-h illumination implemented? Moreover, if 12-h photoperiod was implemented from May to August, such conditions were unnatural, hence the question arises why the experiment was performed such way? 

The statement in the Abstract that the impact of the treatment needs further study is a little confusing. Materials and methods section should be improved as well as the figure captions.

The answers should be incorporated into the text of the future manuscript. At the moment it is not explanatory enough to be considered for publication.

Response: We appreciate your valuable comments about our manuscript and we revise it according to your suggestions one by one.

Abstract

1.96%, 33.36% – such accuracy is unnecessary. Approx. 2%, 33% is quite enough and clearer.

 Response: We appreciate your good comments about our manuscript. We have modified it according to your suggestion.

Introduction

The first sentence sounds odd.

Response: The first sentence has been changed to ‘Light not only provide energy for plant photosynthesis, but also regulate plant growth and development as a signal.’.

Materials and methods

Why the stock plants were of Czech origin?

Response: They were rooted cuttings and all of them were from a single genotype. It should be cutting orchard of Norway spruce was established from the seedlings of Czech origin. The shoot cuttings were harvested from a single clone of the cutting orchard. So the stock plant were of Czech origin.

Why R:B:FR was 7:1:1? Was the photoperiod 12-h or longer? From the sentence L106-107 it is unclear (24 hours?!).

Response: The complete plant LED lighting scheme requires not only blue light (450 nm) and red light (660 nm), but also far-red light (730nm). Blue light and red light provide the spectrum needed for photosynthesis, while far-red light controls the entire process from germination to vegetative growth and flowering. Previous studies showed that FR light is effective for improving the yield and quality of plants produced in a plant factory with artificial light and the application of FR light should be considered with the ratio of existing R and B light sources [7]. The optimization parameters of light environment, the ratio of LED red/blue light/far-red light was set as 8:1:1 in the cultivation of horticultural crop lettuce, and the ratio of red/blue light/far-red light was set as 7:1:1 in the cultivation of cucumber seedlings for rational allocation. R:B:FR 7:1:1 was chosen in our study as it has been applied to the production of plant factories to improve the efficiency of plant growth and save energy. .

The cuttings received natural light during the day and were placed under artificial LED light for 12 hours after sunset. The photoperiod is about 24 hours. We modified it to: “The four LED light sources were monochromatic blue light (B, 460 nm), monochromatic red light (R, 660 nm), monochromatic far-red light (FR, 730 nm) and complex light (control, the ratio of R:B:FR=7:1:1). All of the clones were placed under sunlight during the day and then illuminated after sunset for 12 h under LED light (Lian Bang Zhong Ke Electronic Technology Co., Ltd, Shenzhen, China).”

The methods should be more explanative in most cases.

Response: Thank you for your good suggestion. We add the following information in the M&M section:

2.2. How leaf area was measured?

Response: The needles were scanned and analyzed using the WinRHIZO root analysis system (ecotech ecological technology LTD., Canada).

2.4. Please indicate more details.

Response: We add the following information in the M&M section:

“The chlorophyll content of the needles was measured using 6 clones per LED treatment. Chlorophyll was extracted using 95% ethanol in dark conditions for 72 h at 4°C. Absorbance were measured using a UV-1601 ultraviolet-visible (UV-VIS) spectrophotometer (Shimadzu, Tokyo, Japan). The value of chlorophyll a and chlorophyll b were calculated and then add together for chlorophyll content according to the methods described by Porra et al. [35],. The ratio of leaf chlorophyll content to fresh weight was the chlorophyll content/leaf fresh weight ratio.”

2.5. Please indicate more details about protein measurement.

Response: We add the following information in the M&M section:

Photosynthetic enzymes

Needles (0.1 g for each sample) were ground in a mortar with liquid nitrogen to obtain a fine powder that was subsequently mixed with 150 mg polyvinylpolypyrrolidone (PVPP) and extraction buffer (1.5 ml) containing 40 mM Tris-HCl buffer solution (pH 7.6), 10 mM MgCl₂, 0.25 mM EDTA, 5 mM GSSG and silica sand. The homogenate was centrifuged for 15 min at 20,000 g and 4°C, stored at -70°C, and subsequently used to determine the enzyme activity. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was determined as the rate of NADH decline (ε = 6.22 mM^-1 cm^-1) at 340 nm. A 1 mL reaction system containing 40 mM Tris-HCl buffer solution (pH 7.8), 12 mM MgCl₂, 0.4 mM EDTA-Na₂, 1 mM NADH, 10 mM ATP, 10 mM phosphocreatine, 40 mM NaHCO₃, 20 U creatine phosphate kinase, 20 U phosphoglycerate kinase, 20 U phosphoglyceraldehyde dehydrogenase, 5 mM RuBP, and 100 μL enzyme liquid was initiated upon addition of RuBP. The results were recorded in alternating intervals of 10 s, and the drop-out absorbance value at 230 nm within 3 min was determined.

PEPC activity: The phosphoenolpyruvate carboxylase (PEPC) reaction was coupled with the malate dehydrogenase reaction and assayed at 30°C to monitor the reduced form of nicotinamide-adenine dinucleotid (NADH) oxidation at 340 nm using a Gilford recording spectrophotometer. According to the instruction of PEPC kit (Solarbio, 50T) and refer to the previous study [36], the PEPC activity were analyzed and calculated.

The ratio of PEPC to Rubisco was the PEPC activity/Rubisco activity ratio.

2.7. Please provide more details about the chlorophyll fluorescence measurement protocol to allow the study to be reproducible (temperature, light-saturating pulse, actinic light, saturating pulses etc.).

Response: We add the details about the chlorophyll fluorescence measurement protocol.

Four samples were selected from the same clone to measure the chlorophyll fluorescence parameters for each treatment. In a clear and windless day, a row of needles (8-10 needles) of lateral branches (three lateral branches for each cutting and six cuttings per LED treatment) were glued with transparent glue. The middle part of the leaves was clamped with a dark adaptor. After dark adaptation for 30 min, the probe was placed on the clip. The needles were exposed to saturated pulse light (3000 mmol/m²/s¹) for 1 s. Initial fluorescence (Fo) and maximum fluorescence (FM) of dark adaptation were read directly from the instrument, and the variable fluorescence (Fv) was calculated, Fv = Fm - Fo, PSII maximum light energy conversion efficiency Fv/Fm. Two intermediary steps designated J and I appeared at 2 and 30 ms, respectively; hence, a fast rise of the chlorophyll a fluorescence transient with the notation of O-J-I-P was obtained. 

The chlorophyll a fluorescence transients were analyzed by utilizing the original data from the polyphasic fluorescence transients according to JIP test. The following fluorescence parameters were calculated using the JIP test [37]: the quantum yield of electron transport beyond Q_A⁻ (primary quinone) (ϕEo), ϕEo=ETo/ABS, where ABS is energy flux for absorption and ET is energy flux for electron transport; the efficiency that a trapped exciton moves an electron into electron transport chain beyond Q_A⁻ (ψ_o), ψ_o=ETo/TRo, where TR stand for energy flux for trapping; the normalized relative variable fluorescence at the K step (W_k), W_k=(F_k-F_o)/(F_J-F_o); the relative variable fluorescence at any time (Vt), Vt=(Ft-Fo)/(Fm-Fo).

Statistics: please indicate the probability level for the Duncan test.

Response: We add ‘p<0.05’. 

Results

Figure captions: please indicate the meaning for B, R, FR, M.

Response: We add the following information: “monochromatic blue light (B, 460 nm), monochromatic red light (R, 660 nm), monochromatic far-red (FR, 730 nm) light and complex light (M, the ratio of R:B:FR=7:1:1)” in the figure legends.

Tables

Table 4: the values should be presented as 0.XXX to indicate all significant figures. At the moment, the results of the statistical test seems illogic in the case of : Fv/Fm.

Response: Thank you for your valuable suggestion. We modify the values for retaining three significant digits.

Discussion

Based on the literature and the questions indicated in my general remarks, try to emphasise the novelty and innovativeness of your research.

Response: Thank you! We modified the discussion. We included how the supplemental red light might influence the phytochrome photostationary state (PSS) in seedlings and how these results can contribute to our knowledge on physiological performance of Norway spruce seedlings in plantations, in forest understory etc.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The ms has improved from the its 1st version, the M & M now describing much better the material used and methods in more detail. Also presentation of results is now more consistent and easier to follow.

My only comment concerns the conlusions and the same matter in the discussion. The sentence "Basing on the results, it may promote the regeneration of spruce forest under canopy, and accurately improve the forest quality by thining and pruning" (r709-711). It is difficult to understand to which "it" in this sentence refers.  Seems to refer to "light spectra" in the previous sentence, but does it then make sense ? How does light spectra improve the forest quality by thinning or pruning ?? Or do you mean that improving light conditions under canopy by thinning or pruning, one could expect to improve forest quality???  Please either revise or remove.. There is the same difficulty to undertand the actual Discussion text on this same matter (r560-561), so please revise it too.

Checking the English language of the revised parts of text  would still be desirable.

Author Response

Response: Thank you for your good suggestion. We remove the sentence "Basing on the results, it may promote the regeneration of spruce forest under canopy, and accurately improve the forest quality by thinning and pruning" (r709-711), and the sentence “Basing on the results, it may promote the regeneration of spruce forest under canopy, and accurately improve the forest quality by thining and pruning.”.

 Also, we check and polish the English language for the text. Thank you for your valuable suggestion again. 

Reviewer 3 Report

I accept all explanations and alterations to the manuscript which improve its scientific soundness.

Author Response

Response: We check and polish the English language for the text. Thank you very much for your valuable suggestion again!