Constitutive and Cold Acclimation-Regulated Protein Expression Profiles of Scots Pine Seedlings Reveal Potential for Adaptive Capacity of Geographically Distant Populations
, Monika Sirgėdienė 2, Perttu Haimi 1, Inga Tamošiūnė 1 and Darius Danusevičius 2
Round 1
Reviewer 1 Report
The manuscript provides insights into adaptation due to comparative proteomics analysis of conifer seedlings. The manuscript is well-written and easy to read. The resultsare clearly presented. My only comment is about the presentation of the experiment which it is not very clear.
My suggestion are indicated in the accompanied document.
Comments for author File: 
Comments.pdf
Author Response
We are grateful to reviewer for the comments and suggestions. The manuscript was modified according to reviewer suggestions. English language spelling was corrected as indicated by Markup in the attached Word doccument.
Line 17-21: This sentence is too long and confusing. Split it.
The sentence was modified (lines 19-23): “Protein expression analysis is capable to depict molecular events closely linked to phenotype formation. Therefore, in this study, we used comparative proteomics analysis to differentiate Scots pine genotypes originating from geographically distant populations in Europe, which show distinct growth and cold adaptation phenotypes.”
Line 25-29: The same and here. Try to avoid so long sentences. It is difficult for the reader to follow them
The sentence was modified (lines 29-34): “Acclimation-induced protein expression patterns suggested the presence of a similar cold adaptation mechanism among the populations. Variation of adaptive capacity among the genotypes was potentially represented by a constitutive low level of expression of the Ser/Thr-protein phosphatase, the negative regulator of the adaptive response. Also, overall less pronounced acclimation-induced response in seedlings from the Spanish population was observed.”
Line 34: It is better not to use keywords that already exist in the title
The keyword “cold acclimation” was removed.
Line 60: Here you refer for the first time the target species. It is good to write also the scientific name.
The scientific name of Scots pine was included on line 81
Line 70: It can be a new paragraph.
The paragraph was split on line 95.
Line 99: Refer the dimension of the plastic boxes
Dimensions of the containers were provided on line 148.
Line 101: Did you select this conditions for a specific reason?
Long-day photoperiod was used to promote active growth of seedlings.
The sentence on line 151-153 was modified to include explanation about use of growth conditions: “Constant temperature of 24 °C, 100% humidity and a long-day (16h, 30 µmol m-2 s-1; fluorescent light 5500 K photoperiod was used to promote active growth.”
Line 104: Are there one container for each population? It is not very clear your experimental conditions
Two containers were used for each population. The sentence on line 155-157 was modified to include the information.
Line 105: How many seedlings did you have finally in each box.
Approx. 100 seedlings per container. The sentence on line 159 was modified to include the information.
Line 106: All the seedlings were exposed to the cold acclimation? because below you refer to acclimate and non-acclimate seedlings.
Samples for the active growth experimental group (approx. half of the seedlings) were collected before the cold acclimation treatment. The sentence on line 159-164 was modified to address the ambiguity.
Line 113: each experimental group? What do you mean? Line 117: the experimental groups were the acclimate and non-acclimate seedlings? It is not clear
The experiment included four biological repeats for the active growth and acclimated experimental groups of each population (total 24 samples). Therefore the text should read “four biological repeats for the active growth and acclimated experimental groups of each population”.
The error was corrected on line 169 and 174.
Author Response File: 
Author Response.doc
Reviewer 2 Report
Research entitled " Constitutive and Cold Acclimation-regulated Protein Expression Profiles of Scots Pine Seedlings Reveal Potential of Adaptive Capacity of Geographically Distant Populations " present an important scientific and economic topic of scots pine adaptation to changing environmental conditions.In the research, authors descraibe adaptative features on cold diffrent pine genotypes. In the research used sophisticated labolatory and statystic methods which analyzed protein polymorphism resulting from gene expression. The presented laboratory tests and statystical analyzes are most valuable component of the publication. The results obtained will interest a large numer of readers and probably will be cetated in many studies. On the other hand, the small numer of studied population, which does not exclude research, but should to mobilize authors in more accurate literature analysis in the context of climate impact on pine’s adaptive features. The authors, when analyzing the adaptive features of pine, did not include the analysis of climate variables differentiating the studied populations. Summarizing, in publication lacks analysis of the effect of temperature (cold) on pine adaptation. Specific comments were made in this context:
line 41 – genetic mechanisms incliuding epigenetic mechanisms are not described in sufficient detail.
Line 44 – what are the adaptive conseqences of these genetic features.
Line 90-93 – for many reasons, the autochthonousness of the studied populations should be thoroughly discussed in the publication. In addition, we know nathing about the orgin of the populations (natural, artificial afforestation).
Line 284 – 287 – not much is known about the differing environmental conditions of the studied populations.
Author Response
We are grateful to reviewer for the comments. The attached manuscript was modified to include reviewer suggestions.
line 41 – genetic mechanisms incliuding epigenetic mechanisms are not described in sufficient detail.The first paragraph (lines 50-65) was expanded to include references to previous research about genetic and epigenetic basis of adaptive capacity in forest trees.
Line 44 – what are the adaptive conseqences of these genetic features.The introduction on lines 98 through 105 was expanded to elaborate on the cold induced physiological and gene expression response in pine or coniferous trees.
Line 90-93 – for many reasons, the autochthonousness of the studied populations should be thoroughly discussed in the publication. In addition, we know nathing about the orgin of the populations (natural, artificial afforestation).The populations included in the study were native and naturally regenerating over many generations. More detail information about geographical location and climatic zones of the populations was added on lines 130-140:
“We used seeds of Scots pine collected from the three autochthonous populations representing the Mediterranean, temporal and boreal climatic zones in Europe: central Spain (Valsain Forest mountains, Sierra de Guadarrama prov., 40°49‘ lat., 3°57‘ long., 1828 m. a.s.l. alt.), central Lithuania (Kazlu Ruda forest tact, 54°46‘ lat., 23°32‘ long., 79 m. a. s. l. alt.) and northern Finland (Suomussalmi forest, 65°07‘ lat., 28°49‘ long., 233 m. a. s. l. alt.), respectively (Figure 1). The seeds were collected in native Scots pine stands naturally-born over generations in large forest tracts, where influence of non-autochthonous sources is negligible. These populations mainly represent a geographically large latitudinal gradient of genepools adapted to distinct photoperiodic and temperate requirements to initiate dormancy and cold hardiness processes that have a major effect on timing of cold acclimation of the seedlings.”
Line 284 – 287 – not much is known about the differing environmental conditions of the studied populations.Additional information was provided as described in the comment # 3.
Author Response File: Author Response.doc
