Advances in Barley Breeding for Improving Nitrogen Use Efficiency

Round 1

Reviewer 1 Report

Chen et al. reviewed breeding efforts in improving barley NUE in the manuscript. NUE is a complex trait which controlled by multiple QTL, and contradictory conclusions may be drawn due to differential quantification methods. Advances in this direction have been impeded by lack of effective methodology for phenotype evaluation. The knowledge gained from NUE in cereals has been scarce, as can be seen from this manuscript. There is an urgent need to explore molecular mechanisms underlying superior NUE particularly under the global climate change. The manuscript presented a collection of QTL mapped, transporters tested, quantification protocols being used, varieties with enhanced NUE, and new bioengineering technologies. Thus, this manuscript may be of interest to cereal researchers, inspiring new studies to achieve breakthrough in this recalcitrant NUE trait.

Assisted with big-data analysis and modern technologies, phenomics has been emerged as a powerful tool to acquiree high-dimensional phenotypic data on both organism-wide and field population-wide scales. I wonder if (and how) NUE has been evaluated by phenomics, and if any progress is made. More discussions in this regard can be added to the section 7 (Challenges and Perspectives).

I would like to suggest authors to describe the molecular mechanisms underlying the enhanced NUE by transporters and other cloned genes. I totally understand if this is beyond the author’s research background.

Some minor comments are listed below.

1.       Suggest to simplify lines 38-41, such as “causing environmental problems and escalating global warming”  

2.       Line 43, please change “873 m3 of”

3.       Lines 50-51 and probably otherwhere. “NUE comprises two distinct processes:” which does not make sense. How about “NUE is determined by efficiencies of two distinct processes, N uptake and N utilization (NUpE and NUtE)”?

4.       Please simplify lines 87-100. It’s an odd reading.

5.       Repetitive “NUE or low-N tolerance”

6.       Line 159, delete “in” from “in recently”

7.       Lines 186-187, please revise.

8.       Lines 204-209, please revise.  

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 1, 

Thanks for your detailed and constructive suggestions. We have revised the manuscript according to your comments . Please see below detailed revision notes. The revised parts in the text are marked in red font.

Best,
Zhiwei

Chen et al. reviewed breeding efforts in improving barley NUE in the manuscript. NUE is a complex trait which controlled by multiple QTL, and contradictory conclusions may be drawn due to differential quantification methods. Advances in this direction have been impeded by lack of effective methodology for phenotype evaluation. The knowledge gained from NUE in cereals has been scarce, as can be seen from this manuscript. There is an urgent need to explore molecular mechanisms underlying superior NUE particularly under the global climate change. The manuscript presented a collection of QTL mapped, transporters tested, quantification protocols being used, varieties with enhanced NUE, and new bioengineering technologies. Thus, this manuscript may be of interest to cereal researchers, inspiring new studies to achieve breakthrough in this recalcitrant NUE trait.

Response: Agree. One of the most important research aspects is the establishing of methods for evaluation and rapid screening of crop germplasms with high nitrogen use efficiency. Chlorate treatment based identification of nitrogen-efficient crop germplasms and the important progress of cloning and identification of related nitrogen-efficient genes in rice based on this treatment is a good example. Thanks.

Assisted with big-data analysis and modern technologies, phenomics has been emerged as a powerful tool to acquire high-dimensional phenotypic data on both organism-wide and field population-wide scales. I wonder if (and how) NUE has been evaluated by phenomics, and if any progress is made. More discussions in this regard can be added to the section 7 (Challenges and Perspectives).

Response: Added (please see below).

In addition, the high-throughput phenomics is being developed in estamation of responses to nitrogen treatment in crops by using non-invasive imaging systems, spectroscopy, image analysis, robotics, enzyme based sensors, etc [75-78]. These methods can provide a large amount of real-time data, which will have a revolutionary impact on the estimation of phenotypes in the study of NUE in barley, and will also be an important research direction.

I would like to suggest authors to describe the molecular mechanisms underlying the enhanced NUE by transporters and other cloned genes. I totally understand if this is beyond the author’s research background.

Response: Revised.

Compared with rice, the research progress of barley in this area is still relatively limited. We have already described it intensively in section of “4. Quantitative Trait Loci (QTL) Mapping and Gene Cloning Related to NUE in Barley”, but also in several other sections. Anyway, we have provided further descriptions of the situations of these genes in the section 4. I am not sure if you are happy with this revision (please see below).

Substantial progress has been made in gene identification for improving NUE in other crops, especially in rice [23, 24], and this has been reviewed by Li et al [54]. In bar-ley, however, although many potential candidate genes have been predicted, their real functions have not yet been identified [32, 41]. There are a few reports about identified N-efficiency genes in barley, such as one describing a barley gene encoding alanine aminotransferase (HvAlaAT) that significantly improved NUE in rice [55,56], and an-other two recent reports showing that HvNRT2.1 and HvNLP2 might also be benefit for improving the NUE [39, 57]. It was showed that the biomass and grain yield of HvAlaAT overexpression transgenic rice lines were significantly higher than sibling nulls or wild-type controls under different N application rates. The seeds of transgenic Arabidopsis thaliana overexpressing the HvNRT2.1 gene were significantly enlargered, including seed length and width, resulting in increased thousand-kernel weight. In the hvnlp2 mutants, not only the expressions of the nitrate-responsive genes were sup-pressed under nitrate treatment, but other traits were also inhibited, such as biomass, seed yield and NUE. It suggested that the HvNLP2 gene might play an important role in improving NUE.

Some minor comments are listed below.

1. Suggest to simplify lines 38-41, such as “causing environmental problems and escalating global warming”  

Response: Accepted (please see below).

In addition, the overall efficient use rate of N fertilizers is still no more than 50%, and up to 75% of applied N is lost by leaching into the soil, causing environmental problems and escalating global warming [5–8].

2. Line 43, please change “873 m3 of”

Response: Revised.

3. Lines 50-51 and probably other where. “NUE comprises two distinct processes:” which does not make sense. How about “NUE is determined by efficiencies of two distinct processes, N uptake and N utilization (NUpE and NUtE)”?

Response: Accepted (please see below).

NUE is determined by efficiencies of two distinct processes, N uptake and N utilization (NUpE and NUtE) [1,10,11].

4. Please simplify lines 87-100. It’s an odd reading.

Response: Rephrased (please see below).

De et al found that many barley traits were affected by different levels of N fertilizer application at the flowering stage, including chlorophyll content, net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), initial fluorescence (Fo), maximum fluorescence (Fm), maximal photochemical efficiency (Fv/Fm), photochemical quenching coefficient (qP), N accumulation rate after anthesis, contribution rate of N accumulation to grain after anthesis, leaf N translocation rate, stem N translocation rate, contribution rate of leaf N translocation to grain, contribution rate of stem N translocation to grain, N fertilizer production efficiency (NGPE) (NGPE=Grain yield/N accumulation in above ground of barley plants), N agriculture efficiency (NAE) (NAE=(Grain yield with N fertilization–Grain yield without N fertilization)/N fertilization application), N fertilizer partial productivity (PEP) (PEP=Grain yield/N fertilizer application), and N physiological efficiency (NPE) (NPE=(Grain yield with N fertilization-grain yield without N fertilization)/( N accumulation in above ground of barley plants with N fertilization- N accumulation in above ground of barley plants without N fertilization)).

5. Repetitive “NUE or low-N tolerance”

Response: Revised.

6. Line 159, delete “in” from “in recently”

Response: Deleted (please see below).

Recently, Karunarathne et al [34] just established a method for identification of barley varieties with different NUE according to leaf chlorosis by the combined treatment of low-N and chlorate.

7. Lines 186-187, please revise.

Response: Revised (please see below).

“Saal et al [42] detected 82 QTL for agronomic characteristics under two N fertilization conditions in six different environments.”

8. Lines 204-209, please revise.

Response: Revised (please see below).

With the development of next generation sequencing technology and its greatly reduced cost, QTL mapping based on SNP markers has become very convenient, and it can be accomplished by re-sequencing of DNA from two bulked DNA pools [46].

Reviewer 2 Report

I premise that this is not a merit value of the submitted manuscript; I only verify that the manuscript Karunarathne et al 2020 published in the same journal covered many sections included also in your manuscript. I do not retain two-years a sufficient time to significantly improve the knowledge on NUE for barley. I briefly compared the knowledge included in both papers and I confirm my initial feeling.    

Author Response

Dear Reviewer 2,

Thank you very much for reviewing our manuscript. We have made some responses to your comments. Please see below.

Best,
Zhiwei

I premise that this is not a merit value of the submitted manuscript; I only verify that the manuscript Karunarathne et al 2020 published in the same journal covered many sections included also in your manuscript. I do not retain two-years a sufficient time to significantly improve the knowledge on NUE for barley. I briefly compared the knowledge included in both papers and I confirm my initial feeling.  

Response:  Our perspective is different from theirs although some aspects are overlap. They are less involved in some aspects, such as the research on NUE phenotyping, plant-microbe interaction for improving NUE,  crop breeding for high NUE. In addition, their team published two latest articles on GWAS and chlorate-based NUE phenotyping in barley just after this review, not to mention the work of other teams, there are still relatively big changes. Thank you very much.

 

Reviewer 3 Report

The manuscript is well written and the subject is of interest for the scietific community.

Minor revisions:

Improve quality of figure 1

A section in which is discussed comparison with aothers cereals shluold be added

Author Response

Dear Reviewer 3, 

Thanks for your detailed and constructive suggestions. We have revised the manuscript according to your comments . Please see below detailed revision notes. The revised parts in the text are marked in red font.

Best,
Zhiwei

The manuscript is well written and the subject is of interest for the scietific community.

Response: Thank you very much.

Minor revisions:

Improve quality of figure 1

Response: Improved.

A section in which is discussed comparison with other cereals should be added

Response: We have introduced some important progress in other cereals in section “7. Challenges and Perspectives”, especially in rice, and these progress are benefit for the research on NUE in barley, and some of them have already been started. Please see the revised version of manuscript. Thanks.

Round 2

Reviewer 2 Report

I did not find both the answer and the revisions adequate to change my opinion. I remark again that it is not a merit judge on the manuscript but I retain that the same journal have recently published a manuscript rather similar.