Effects of different nitrogen fertilizers on two wheat cultivars: An integrated approach

Abstract Investigation of cultivated plant physiology grown under low energy input plays an important role to indicate their fitness to the new environmental conditions. The durum‐wheat cultivars Creso and Dylan were tested to evaluate the growth, production, and proteomic and transcriptomic profiles of the crop under different synthetic and organic nitrogen fertilization regimes. In this work, a two‐dimensional gel electrophoresis (2‐DE) approach combined with liquid chromatography–mass spectrometry (LC–MS) was used to investigate the protein changes induced by the use of different nitrogen sources (hydrolysate of proteins 1 and 2, rhizovit, synthesis, leather) on wheat plants. Proteomic studies were integrated with qPCR analysis of genes related to glutamine synthetase/glutamine‐2‐oxoglutarate aminotransferase (GS‐GOGAT) and tricarboxylic acid (TCA) metabolic pathways because most relevant for nitrogen‐dependent plants growth. The proteomic analysis lead to the isolation of 23 spots that were able to distinguish the analyzed samples. These spots yielded the identification of 60 proteins involved in photosynthesis, glycolysis, and nitrogen metabolism. As an example, the quinone oxidoreductase‐like protein and probable glutathione S‐transferase GSTU proteins were identified in two spots that represents the most statistically significant ones in Dylan samples. Transcript analysis indicated that related genes exhibited different expression trends; the heat map also revealed the different behaviors of the hydrolysates of the proteins 1 and 2 nitrogen sources. The effects of nitrogenous fertilizers at the proteomic and agronomic levels revealed that plants fertilized with synthesis or rhizovit gave the best results concerning yield, whereas rhizovit and protein hydrolysates were most effective for proteins content in the grain (% of dry weight). Therefore, all parameters measured in this study indicated that different kinds of nitrogen fertilization used have a relevant impact on plant growth and production.


Title
and abstract have been modified as suggested by the Reviewer. Discussion and results have been reformulated as indicated by the Reviewer. In particular, some parts of the discussion were transferred into the results some section, and a small final part has been added.
Other issues.
1) A certain level of variability between the two years of analysis is conceivable, which could be linked to the different climatic conditions that occurred between the two agricultural years considered. In particular, some spots in the gels analyzed during the second year had higher normalized volume levels than those obtained during the first year of analysis. . Despite these premises, some clarifications are necessary: -the three biological replicates analyzed came from three distinct pools, one for the first analysis and two for the second year.
-The spots considered as differentially expressed had a low level of intra-group variance (i.e. between the two years of analysis considered for each sample). For this reason, all the spots that showed a high level of variability between the two years analyzed were not selected. -The differences between the two years considered have had a minor impact on the normalization of the data, as the overall average volume data (optical density) were comparable between the replicates of each sample as well as between different samples.
2) The proteomics results were not validated by qPCR because the identified proteins belonged to metabolic pathways often unrelated to each other. For this reason, we decided to focus on the GS-GOGAT metabolism, believing that such genes could be more influenced by the different forms of nitrogen available. Such an approach has been clarified in the main text as well, as requested.
3) The modulation in the metabolic response highlighted by the proteomic and transcriptomic analyzes show that samples differed more according to the cultivar they belong to and, to a lesser extent, depending on the applied treatment (i.e. type of fertilizer used). If we consider the production data, synthetic fertilizers (Urea, Ammonium Nitrate, Rhizovit) have given the best production performances. In the same way, proteomic data show how the treatments with synthetic fertilizers generically induce an upregulation in the two cultivars considered. By way of example, the data reported in spots 15 and 17 show the most significant differences. The Probable glutathione S-transferase GSTU1 protein, identified in spot 17, is an ideal candidate, as the expression of this protein class is influenced, both in the leaves and in the root, under conditions of nitrogen deficiency. Regarding the transcriptomic data, the results obtained are strongly influenced by the type of cultivar considered. As for the Creso cultivar, the CS (Creso synthesis) sample has numerous upregulated genes, followed by the CL (Creso leather) sample. The overall trend concerning the Dylan cultivar is different, where a high number of induced genes is reported in the samples treated with protein hydrolysates (DHP1, DHP2) and, to a lesser extent, in the sample treated with leather (DL). From the physiological point of view, these results could be explained by specific characteristics related to the two cultivars, and in particular to the form of preferentially absorbed nitrogen (nitric or ammoniacal). In fact, the fertilizers used, except synthetic products (Ammonium Nitrate and Rhizovit, Supplemental Table 1) do not contain any forms of ammonia nitrogen but contain nitrogen exclusively in the nitric form.
The paragraph above has been introduced in the Discussion section of the revised manuscript.
Reviewer 2: 1. Abstract has been modified according to the Reviewer's indications.
2. Some parts of the text have been deleted (see changes highlighted version of the manuscript), Table 1 of the original version of the manuscript has been included in the new Figure 1, while Table 2 and 6 of the original version have been moved to Supplemental data, as suggested. Figures 5 and 6 have not been merged, since the integration of the two images would lead to some problems of legibility (too small characters). 4. As now specified in the discussion (line 596), the differences between the two cultivars about the expression of the nitrate transporters could be linked to the form of nitrogen preferentially absorbed by the cultivar (nitric or ammonia form). In the work of Curci et al., 2017 the effect linked to the nitrogen deficiency in the Svevo cultivar was evaluated, using a predominantly molecular approach. In this case, the results show that high-affinity transporters, and in particular NRT2.5, are upregulated in the roots, while no phenomena of up-or downregulation in the leaves have been highlighted. The Svevo cultivar is a relatively recent cultivar (1996) and has characteristics comparable to the Creso cultivar.
5. Some Authors recently stated that the current understanding of N effects on photosynthetic electron transport rate and partitioning, as well as its impact on photosynthesis under [CO2]e, is inadequate. (Zhang X.C., X. F. Yu, Y. F. Ma, Effect of nitrogen application and elevated CO2 on photosynthetic gas exchange and electron transport in wheat leaves. Photosynthetica, vol 51, 593-602, 2013); however according to the data reported in the paper, they concluded that sufficient N improved light energy utilization in wheat flag leaves thus benefiting to photosynthetic assimilation.
Also Zhou Y-h et al. (Effects of nitrogen form on growth, CO2 assimilation, chlorophyll fluorescence, and photosynthetic electron allocation in cucumber and rice plants. J Zhejiang Univ Sci B 12:126-134, 2011) found that the N source had little effect on photosynthetic electron allocation in rice plants, except that NH4 + -grown plants had a higher O2-independent alternative electron flux than NO3 -grown plants. NO3 reduction activity was rarely detected in leaves of NH4 + -grown cucumber plants but was high in NH4 + -grown rice plants. These results demonstrate that significant amounts of photosynthetic electron transport were coupled to NO3 assimilation, an effect more significant in NO3 -grown plants than in NH4 + -grown plants.