Response to Reviewers' comments:

Reviewer #1:
The submitted manuscript "NADP-dependent enzymes are involved in salt and
hypoosmotic stress in cucumber" deal with the osmotic effects and salt on
physiological parameters and enzymatic activities in an economically important plant
species. The suggested connection of NADPH/NADP ratio with plant metabolism is
on interest as it could be one of the mechanism of the stress response in plants.
However, the presented manuscript needs substantial revision for publication. In
general, the experimental design and the interpretation of data do not fit very well
together, making the “main message” of the article rather unclear for the reader. The
authors are comparing the plants grown in pots (control) with the plants treated in
hydroponics. In such case the plants have to respond to a mixture of different effects
such as transfer from pot to hydroponics (that are quite different conditions for the
roots e.g. oxygen availability, mucilage on the root surface etc.), osmotic shock (hypoor hyper-) and salt. Moreover, there may be some effect of nutrients-lack for 3 days (in
pots were sure present some nutrients although no any data about them is available in
Material and Methods, whereas in hydroponics were the plants grown on distilled
water only, with or without NaCl). Therefore there is a big question if plants in pots
are a good control. Maybe statistical comparison of salt treatment with water
(“hypoosmotic stress”) would be also informative as well as the time-course of the
response. The authors should be careful in interpretation of their data and try to
explain better what they really observed (e.g. what is likely related to salt treatment
and what to osmotic effects).
Authors: We are aware that the effect of stress on the plant is a complex process. The
reviewer is right that out experimental design involved also other types of stress such
as mechanical stress, oxygen availability, and nutrient stress. To minimize nutrient
stress or mucilage on the root surface the experiment was carried out only as shortterm. We endorse that these stresses would not be a part of salt soil experiment. On the
other hand, experimental induction of salt stress in the soil brings also difficulties, e.g.
distribution of salt solution in the soil and related apparent soil electrical conductivity
are influenced by a combination of physico-chemical properties including soluble salts
(it is the same with culture medium), soil matter content, bulk density, organic matter,
clay content and mineralogy and soil temperature (Corvin and Lesch, 2005). All these
factors influenced salt effect on plants and we wanted to be sure that plants were
influenced by specific given concentration of NaCl (100 mM). We agree that influence
of distilled water could also serve as a control eliminating the effect of above
mentioned stresses. Therefore we add a table with statistical comparison of salt
treatment with distilled water and particular outputs are complemented in the Results
and Discussion section. We believe that we bring new information about metabolic
processes in plants under described conditions. Mechanic stress, oxygen availability
and possible nutrition stress is equal in distilled water and 100 mM NaCl, the
comparison can show the only effect of salt.
Supplementary Table 1 Statistical comparison of plants exposed to 100 mM NaCl
(salt) with plants in distilled water (water) and with plants grown in soil (soil).
Pearson´s coefficients are presented. Symbols ↑, ↓ and N indicate a decrease, an
increase or no change of particular parameter. ND means not determined.
This Table could be a part of the MS or supplement if the reviewers and editor wish.
Day of stress
RWC/Fig.1
Na+ leaf /Fig. 2A
Na+ root/Fig. 2B
K+ leaf/Fig. 2C
K+ root/ Fig. 2D
Phenolics/Fig. 3A
Flavonoids/Fig.
3B
Rubisco/Fig. 4
Proteins leaf Fig.
5A
Proteins root Fig.
5B
NADP leaf Fig.
7A
NADPH leaf Fig.
7B
NADP root Fig.
7C
NADPH root Fig.
7D
G6PDH leaf Fig.
8A
NADP-ICDH leaf
Fig. 8B
NADP-ME leaf
Fig. 8C
SDH leaf Fig. 8D
NP-GAPDH leaf
Fig. 8E
G6PDH root Fig.
9A
NADP-ICDH root
Fig. 9B
NADP-ME root
Fig. 9C
SDH root Fig. 9D
Glucose-1-DH
leaf Fig. 10A,B
Gluconate-2-DH
leaf Fig. 10A,B
Ribose-1-DH leaf
Fig. 10A,B
Galactose-1-DH
leaf Fig. 10A,B
Glycerol-2-DH
leaf Fig. 10A,B
Glucose-1-DH
root Fig. 10C,D
Gluconate-2-DH
root Fig. 10C,D
Ribose-1-DH root
Fig. 10C,D
Galactose-1-DH
root Fig. 10C,D
Glycerol-2-DH
root Fig. 10C,D
Salt/
0
Salt/
Water/
Salt/
1
Salt/
Water/
Salt/
2
Salt/
Water/
Salt/
3
Salt/
Water/
soil
↓0.002
N0.548
↑0.001
N0.284
N0.875
↑0.043
↑0.031
water
↓0.002
N0.850
↑0.005
N0.560
N0.244
N0.137
N0.089
soil
N0.905
N0.297
N0.706
N0.445
N0.483
↑0.021
N0.317
soil
↓0.002
↑≤0.001
↑0.003
N0.196
↓0.009
↑0.036
N0.062
water
↓0.005
↑≤0.001
↑0.007
N0.814
N0.194
↑≤0.001
↑0.042
soil
N0.300
N0.082
N0.101
N0.200
N0.833
↓0.038
N0.725
soil
↓≤0.001
↑≤0.001
↑0.004
N0.347
↓≤0.001
↑0.003
↑0.002
water
↓0.002
↑≤0.001
↑0.026
N0.041
↓0.005
N0.475
N0.051
soil
N0.420
N0.469
N0.132
N0.077
N0.141
↑0.001
↑0.001
soil
↓≤0.001
↑0.002
↑≤0.001
↓0.038
↓0.001
↑0.001
↑0.005
water
↓0.017
↑0.003
↑0.011
N0.168
↓≤0.001
↑0.006
↑0.033
soil
↓0.004
N0.497
N0.154
N0.504
N0.329
N0.064
N0.614
N0.865
N0.132
N0.184
N0.705
N0.191
↑0.003
N0.365
N0.060
N0.091
↑≤0.001
N0.250
↓0.003
↓0.023
↑0.012
↓0.004
↑0.009
N0.089
N0.344
↓0.005
N0.130
↓0.003
↑≤0.001
N0.652
↓≤0.001
N0.148
N0.072
N0.409
N0.218
↑0.017
↓0.032
N0.108
N0.791
N0.103
N0.925
N0.824
N0.652
↓0.035
N0.536
↓0.008
↑0.017
↑0.003
N0.523
↑0.035
↑0.006
↓0.004
↑0.016
↑0.015
N0.267
N0.844
N0.638
↓0.017
N0.069
N0.209
↓0.034
N0.330
N0.061
N0.130
N0.109
N0.650
N0.267
N0.962
N0.484
N0.609
N0.156
N0.372
N0.900
N0.065
N0.053
N0.365
N0.151
↓0.026
N0.148
N0.998
N0.099
N0.201
N0.912
N0.897
N0.938
N0.186
↓0.040
↑0.018
N0.858
↓≤0.001
↑0.007
N0.370
↑0.030
↓0.029
N0.179
↓0.005
↑0.022
N0.056
N0.998
↑0.022
↑0.002
↓0.009
↑0.001
↓≤0.001
↓0.009
↓0.013
N0.149
↓0.004
↑0.005
N0.271
↓0.034
↑0.044
↑0.009
N0.130
↑0.047
↓0.046
N0.681
↓0.021
N0.061
↓0.006
↑0.023
N0.087
N0.126
N0.456
↑0.002
↓0.006
↑≤0.001
N0.511
↓0.002
↑0.003
↓0.009
↓0.002
N0.149
N0.409
N0.182
↓0.015
N0.089
↑0.030
N0.157
↓0.012
N0.623
↓0.021
N0.161
N0.611
N0.196
N0.158
↑0.009
↓0.004
N0.080
↑0.001
↑0.016
↑0.003
↑0.002
N0.118
↓0.007
↓0.010
N0.346
↓≤0.001
↓0.001
N0.084
↓≤0.001
↓≤0.001
↑0.001
N0.750
N0.563
N0.354
N0.569
N0.848
N0.780
N0.773
↓0.017
↑0.023
↓0.003
↓≤0.001
↑0.003
↑0.007
N0.233
↑0.022
N0.373
N0.081
N0.150
N0.366
↓0.020
↑0.010
N0.238
↓≤0.001
↑≤0.001
↑0.006
N0.213
N0.079
N0.055
↑0.037
N0.230
N0.817
N0.836
↓0.020
↑0.021
↑0.020
↓0.008
↓0.017
ND
↓0.019
ND
N0.327
ND
↓0.018
ND
↓0.002
ND
↑0.007
ND
N0.197
↑≤0.001
↓0.049
N0.610
N0.269
N0.863
ND
ND
ND
ND
ND
ND
↑0.004
↑0.002
N0.631
↑≤0.001
↑0.015
N0.180
ND
ND
ND
ND
ND
ND
↑0.005
↑0.005
N0.544
N0.063
N0.070
N0.588
ND
ND
ND
ND
ND
ND
N0.418
N0.537
N0.202
↑0.006
↑≤0.001
↓0.001
ND
ND
ND
ND
ND
ND
↑0.014
↑0.031
N0.727
↓0.031
N0.737
↓0.018
ND
ND
ND
ND
ND
ND
↓0.008
↑0.010
↓0.002
N0.600
N0.130
N0.083
ND
ND
ND
ND
ND
ND
↑0.003
↑0.004
N0.407
N0.251
N0.971
N0.487
ND
ND
ND
ND
ND
ND
↑0.032
↑0.039
N0.713
↓0.003
N0.062
N0.092
ND
ND
ND
ND
ND
ND
↑0.002
↑0.004
↑0.016
N0.052
↓0.038
↑0.016
ND
ND
ND
ND
ND
ND
Some of the results are confusing and/or not very well explained in results and
discussion (in details see below). I suggest some revision of the experimental data (for
Figures 1-3). Page 10, line 192: “biological experiments” means biological replicates?
Or the whole experiments were repeated 4-times?
Authors: Biological experiment means newly grown and stressed plant. The samples
for individual assays were collected in triplicates.
Page 13, lines 286-288: The meaning of this paragraph is unclear!
Authors: This paragraph “An accumulation of Na+ ions was higher in roots than in
leaves (Fig. 2), what is probably related to decreased extension of metabolic response
to stress in this tissue maybe due to different regulation than in leaves.” was edited.
We though: Higher accumulation of Na+ ions in roots than in leaves could be a
consequence of decreased defense responses mediated by NADP-depended enzymes
(Fig. 9, day 3). Furthermore, for example G6PDH and maybe also some other NADPenzymes are differently regulated in roots and in the leaves by thioredoxin. With
regard to the reference to Fig. 9, it is also replaced now and simplified, thank you for
valuable notice.
lines 289-290: Discussion is focused to NADPH/NADP ratio, but this ratio is present
in the figures.
Authors: Since the content of NADP in leaves of stressed plants increased and
NADPH didn´t, we decided for separate time course presentation. The ratio can be
estimated from presented data. However, we could add also the Figure with the ratio,
but it would be a presentation of one result 2-times.
Page 14, lines 298-302: According to the Figure 8, there is no any increase of SDH
activity on salt, therefore it can hardly contribute on increase of phenolic of flavonoid
content presented in figures 3A and B.
Authors: But as we indicated: the fresh weight SDH activity was increased in salt
stressed plants (data not shown), therefore the participation of this enzyme on increase
of phenolics is possible. On the other hand, the pathway from shikimate to phenolic
compounds through aromatic amino acids is very complex with different regulation
points and thus directing the metabolic flow can cause the accumulation of phenolics.
Fig. 1: Decrease of RWC on day 3 in plants treated with distilled water is rare. What
may be the reason?
Authors: During time course of hypoosmotic stress, the RWC wasn´t statistically
different from plants growth in the soil (supplementary table) with the only exception
day 3. Maybe a decrease of the plant turgor after 3 days of stress caused the decrease
of RWC. Hypoosmotic stress could affect the plant metabolism, the plant cells were
not able to maintain turgor and water in the cells and thus RWC was decreased (but
not as much as in salt stress).
Fig. 2: This results are really confusing! How can the authors explain the increase of
root Na+ content in the plants treated 1 day with water (Fig. 2B, day 0 versus day1)?
What is it coming from? What is the source of this Na+? The significant increase of
K+in leaves threated with water is also surprising. Especially when it is present only
in day 0 and day 2. It is likely that this is caused by comparison with the plants grown
in pots, which are not very suitable as a control in this case.
Authors: From our point of view, the differences between ion content in plants grown
in soil and in hypoosmotic stress were not so significant, although reviewer is right
that in 4 cases t-test showed that they are, but P coefficient were mostly higher than
0.01. Further analysis showed that there are no differences between both groups in ion
content, which results in revised version of Fig. 2.
Fig. 3: In the case of plants treated with water the results are really inconsistent. This
is the case especially in Fig. 3C. The antioxidant capacity of that plant very high on
day 0 and day 2, but very low on day 1 and day 3 (the description of this results in
page 12 lines 242-243 is not in agreement with the figure). Either the increase of
phenolics only at the day 2 needs some discussion.
Authors: Thank you very much for error notification. Instead of “after 3 days was 3.2fold increased (Fig. 3C)” the sentence should be: after 2 days was 3.2-fold increased (Fig. 3C).
We agree that results concerning antioxidant capacity are inconsistent, therefore we decided
not to present Fig. 3C.
Reviewer #2:
The manuscript “NADP-dependent enzymes are involved in salt and hypoosmotic
stress in cucumber” has been reviewed. The authors examine changes in osmolyte
contents and activities of NADP-dependent enzymes in salt-stressed and
hypoosmotically stressed cucumber plants. The results presented are original and
somewhat new, and could be useful to researchers working on plant stress responses
and/or photosynthesis. However, the current manuscript has many errors and thus
should need revision for publication. Points to be addressed are as follows: 1. There
are so many errors in grammar and word usage (except in the Introduction section).
Because of them, some parts of the manuscript are very difficult to understand. I
would recommend the manuscript be edited by a professional.
Authors: English was edited by a professional.

2. Abbreviations such as RWC, ROS, and PVP are not defined appropriately in the
text. The journal instruction says “Abbreviations in parentheses should be preceded by
the full term when first used (except for those which are very common)”. Similarly, in
the abstract, “RWC” should be spelled out when first used.
Authors: Thank you, all abbreviations (which are not very common) are defined
appropriately in the text now.
3. The information given in the line 198-203 (first paragraph for the Results and
Discussion section) is almost completely redundant with that in the Introduction
section. How about stating in the Introduction section that hypoosmotic stress was
induced by distilled water, and then removing the first paragraph for the Results and
Discussion section?
Authors: You are right. We completely removed this paragraph and stated that
hypoosmotic stress was induced by distilled water in the Introduction section.
Furthermore, we defined controls according to second reviewer.
4. In the line 218-232, it is unclear whether the results regarding K/Na ratio are new or
not. I guess the changes in K/Na ratio in salt-stressed cucumber plants and those in
other plant species have already been characterized in previous studies. Are the results
presented in this study consistent with them?
Authors: Yes, the results presented in this study are consistent with previously
published studies. This phenomenon is known. Relevant section was edited and partly
rewritten.
5. It should be discussed whether Fig. 7 and Fig. 8-10 are consistent. Are the stressinduced changes in NADPH/NADP ratio regulated by any specific NADP-dependent
enzyme, or by many of such enzymes?
Authors: You are right that the consistence of these results were not discussed enough.
It is completed now, thank you. All NADP-enzymes with increased activity can
participate in changes in NADPH/NADP ratio.
6. Is the conclusion (line 360-363) new? In addition, the conclusion seems too strong
to me if it is made only by the findings that NADPH/NADP ratio and activities of
NADP-dependent enzymes are affected by the salt and hypoosmotic stresses.
Authors: The conclusion was rewritten according to comments of both reviewers.
7. In the Fig. 1 legend, replace “relative water content” with “relative water content
(RWC)”.
Authors: The legend was edited, thank you.
8. In Fig. 2, 3, 5 and 7, define D.W. (as dry weight?) in corresponding figure legends.
9.
Authors: The definition of D.W. was completed to these Figs.
In Fig. 2, 3, 5, 7-10, replace “.” with “・”.
Authors: The figures were adjusted.
10. In Fig. 4 and 6, indicate that “55000” and “70000” mean molecular mass or
molecular weight. It would not be good to place these numbers right below the label
“Days of stress”.
Authors: Thank you for this remark, relevant Figs were adjusted.
11. In Fig. 6, define “RU”.
Authors: These dimensionless units were generated by ImageJ program, which use a
graphical method that involves generating lane profile plots, drawing lines to enclose
peaks of interest, and then measuring peak areas (i.e., definite integrals).