Transcriptome and Proteomics Analysis of Wheat Seedling Roots Reveals That Increasing NH4+/NO3– Ratio Induced Root Lignification and Reduced Nitrogen Utilization

Wheat growth and nitrogen (N) uptake gradually decrease in response to high NH4+/NO3– ratio. However, the mechanisms underlying the response of wheat seedling roots to changes in NH4+/NO3– ratio remain unclear. In this study, we investigated wheat growth, transcriptome, and proteome profiles of roots in response to increasing NH4+/NO3– ratios (Na: 100/0; Nr1: 75/25, Nr2: 50/50, Nr3: 25/75, and Nn: 0/100). High NH4+/NO3– ratio significantly reduced leaf relative chlorophyll content, Fv/Fm, and ΦII values. Both total root length and specific root length decreased with increasing NH4+/NO3– ratios. Moreover, the rise in NH4+/NO3– ratio significantly promoted O2– production. Furthermore, transcriptome sequencing and tandem mass tag-based quantitative proteome analyses identified 14,376 differentially expressed genes (DEGs) and 1,819 differentially expressed proteins (DEPs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that glutathione metabolism and phenylpropanoid biosynthesis were the main two shared enriched pathways across ratio comparisons. Upregulated DEGs and DEPs involving glutathione S-transferases may contribute to the prevention of oxidative stress. An increment in the NH4+/NO3– ratio induced the expression of genes and proteins involved in lignin biosynthesis, which increased root lignin content. Additionally, phylogenetic tree analysis showed that both A0A3B6NPP6 and A0A3B6LM09 belong to the cinnamyl-alcohol dehydrogenase subfamily. Fifteen downregulated DEGs were identified as high-affinity nitrate transporters or nitrate transporters. Upregulated TraesCS3D02G344800 and TraesCS3A02G350800 were involved in ammonium transport. Downregulated A0A3B6Q9B3 is involved in nitrate transport, whereas A0A3B6PQS3 is a ferredoxin-nitrite reductase. This may explain why an increase in the NH4+/NO3– ratio significantly reduced root NO3–-N content but increased NH4+-N content. Overall, these results demonstrated that increasing the NH4+/NO3– ratio at the seedling stage induced the accumulation of reactive oxygen species, which in turn enhanced root glutathione metabolism and lignification, thereby resulting in increased root oxidative tolerance at the cost of reducing nitrate transport and utilization, which reduced leaf photosynthetic capacity and, ultimately, plant biomass accumulation.

Seasonal variation of the effect of extremely diluted agitated gibberellic acid (10-30) on wheat seedling development

Objective: Performing a study on a wheat growth bio assay with a homeopathic dilution of gibberellic acid at different seasons of the year. Methods: Grains of winter wheat (Triticum aestivum, Capo variety) were observed under the influence of extremely diluted gibberellic acid (10-30, 30x). Analogously prepared water was used for control. 15 experiments were performed, 9 in autumn season (5 researchers, 4,440 grains per group), and 6 in winter / spring (4 researchers, with 3,140 grains per group). Results: All 9 autumn experiments showed less stalk growth in the verum group (p > 0.01 in 4 cases, p > 0.05 in 3, trend in 2 cases). Mean stalk lengths (mm) were 46.97 + 20.50 for verum and 50.66 + 19.77 for control at grain level (N = 4,440 per group) and + 3.87 and + 3.38 respectively at dish level (217 cohorts of 20 or 25 grains per treatment group). Verum stalk length (92.72%) was 7.28% smaller than control stalk length (100%). In contrast, no reliable effect was found in experiments performed in winter / spring (less stalk growth in 1 case, no difference in 1, more growth in 3 cases). Overall verum stalk length (103.64%) was 3.64% slightly greater than control stalk length (100%). Data were found to be homogeneous within the control groups as well as within the verum groups. Conclusion: Results suggest that especially in the experiments performed in autumn, there was an influence of gibberellic acid 30x on wheat seedling development. The effect size is small when calculation is done on the basis of grains (d = 0.18) but high when done on the basis of dishes (d = 1.02). In contrast, no reliable effect was found in experiments performed in winter / spring. Further experiments should thus be performed in the autumn season.

Pre stimulation by gibberellic acid and the effect of extremely diluted agitated gibberellic acid on wheat stalk growth

Background: In previous multicentre studies[1,2], the influence of a homeopathic ultra high dilution of gibberellic acid on wheat growth was scrutinized. Data showed that this test dilution slowed down stalk growth when experiments were performed in autumn season. Aim: To test the hypothesis that pre treatment of grains with high concentrations of gibberellic acid will enhance the effect of the ultra high dilution of the plant hormone Methods: Grains of winter wheat (Triticum aestivum) were observed under the influence of extremely diluted gibberellic acid (10-30) prepared by stepwise dilution and agitation according to a protocol derived from homeopathy (“G30x”). Analogously prepared water was used for control (“W30x”). Grains (500 or 1000 per group) were pre treated with (not agitated) gibberellic acid 10-5, 10-4 and 10-3 (“Ge-5, Ge-4, Ge-3”) or with water (“W”) for control prior to treatment with G30x or W30x. Seedlings were allowed to develop under standardized conditions for 7 days; plants were harvested and stalk lengths were measured. Results: With regard to pre treatment, it can be seen that the groups treated with Ge-3 showed most growth, followed by the Ge-4 groups, the Ge-5 groups and the non pre treated W groups. This decline is modulated by the application of G30x and W30x (figure 1). The lower the pre treatment concentration of G, the more marked is a slowing down effect of G30x versus W30x. Conclusion: The hypothesis that pre treatment of grains with high concentrations of gibberellic acid will enhance the effect of G30x had to be rejected. In contrast, G30x slowed down stalk growth in the W group with p < 0.001 but only moderately in the Ge-5 and Ge-4 group and not at all in the Ge-3 group. Keywords: homeopathy, wheat stalk growth, gibberellic acid, high dilution References: [1] Pfleger A., Hofäcker J., Scherer-Pongratz W., Lothaller H., Reich C., Endler P.C. The effect of extremely diluted agitated gibberellic acid (10e-30) on wheat seedling development – a two researcher study. Complementary Therapies in Medicine 2011; 19: 164-169. [2] Endler PC, Matzer W, Reich C, Reischl T, Hartmann AM, Thieves K, Pfleger A, Hofäcker J, Lothaller H, Scherer-Pongratz W. Seasonal variation of the effect of extremely diluted agitated gibberellic acid (10e-30) on wheat stalk growth – a multi researcher study. The Scientific World Journal 2011; 11: 1667-1678.

A mechanistic investigation of enhanced nitrogen use efficiency in wheat seedlings after treatment with an Ascophyllum nodosum biostimulant

ABSTRACTReduction in the emissions of the greenhouse gas nitrous oxide and nitrogen (N) pollution of ground water by improving nitrogen use efficiency (NUE) in crops is urgently required in pursuit of a sustainable agricultural future. Utilising an engineered biostimulant (PSI-362) derived from the brown seaweed Ascophyllum nodosum, we examined its effect on wheat seedling growth dynamics and mechanistic spatiotemporal changes at transcriptional and biochemical levels in relation to N uptake, assimilation and NUE. PSI-362-mediated biomass increase was associated with increased nitrate uptake and N assimilation in the form of glutamate, glutamine, free amino acids, soluble proteins and total chlorophyll. Phenotypical and biochemical analysis were supported by evaluation of differential expression of genetic markers involved in nitrate perception and transport (TaNRT1.1/NPF6.3), and assimilation (TaNR1 and TaNiR1, TaGDH2, TaGoGAT, TaGS1). Finally, a comparative analysis of the PSI-362 and two generic Ascophyllum nodosum extracts (ANEs) demonstrated that the NUE effect greatly differs depending on the ANE biostimulant used. In the current context of climate warming the transition of agriculture to a more sustainable model is urgently required. Application and adoption of precision biostimulants creates an opportunity for sustainable crop management, reduced production cost and environmental pollution, while maintaining yields.

A method for obtaining field wheat freezing injury phenotype based on RGB camera and software control

Background Low temperature freezing stress has adverse effects on wheat seedling growth and final yield. The traditional method to evaluate the wheat injury caused by the freezing stress is by visual observations, which is time-consuming and laborious. Therefore, a more efficient and accurate method for freezing damage identification is urgently needed. Results A high-throughput phenotyping system was developed in this paper, namely, RGB freezing injury system, to effectively and efficiently quantify the wheat freezing injury in the field environments. The system is able to automatically collect, processing, and analyze the wheat images collected using a mobile phenotype cabin in the field conditions. A data management system was also developed to store and manage the original images and the calculated phenotypic data in the system. In this experiment, a total of 128 wheat varieties were planted, three nitrogen concentrations were applied and two biological and technical replicates were performed. And wheat canopy images were collected at the seedling pulling stage and three image features were extracted for each wheat samples, including ExG, ExR and ExV. We compared different test parameters and found that the coverage had a greater impact on freezing injury. Therefore, we preliminarily divided four grades of freezing injury according to the test results to evaluate the freezing injury of different varieties of wheat at the seedling stage. Conclusions The automatic phenotypic analysis method of freezing injury provides an alternative solution for high-throughput freezing damage analysis of field crops and it can be used to quantify freezing stress and has guiding significance for accelerating the selection of wheat excellent frost resistance genotypes.

Meta-analysis of the role of zinc in coordinating absorption of mineral elements in wheat seedlings

Background Zinc (Zn) is an important nutrient for human beings, which is also an essential micronutrient for crop growth. This study investigated the role of Zn in coordinating the mineral elements absorption in modern wheat (Triticum aestivum L.) cultivars with a new developed method. Results A method was developed, and showed a robust capability to simultaneously investigate seven mineral elements uptake in wheat seedling. With this method, we found low Zn supply (< 1 μM) promoted the absorption of potassium (K), magnesium (Mg) and manganese (Mn) in wheat seedling, while high Zn supply (> 1 μM) significantly inhibited the absorption of these elements. Cultivars with the green genes (Rht-B1b and Rht-D1b) showed a higher uptake capability on ammonium (NH4+), and cultivars with Rht-B1b allele can uptake more phosphors (P), K, calcium (Ca), Mn and Zn compared to cultivars with Rht-D1b. Further analysis indicated higher uptake capability of NH4+ in cultivars contained Rhts was independent of Zn. Conclusion The key role of Zn in coordinating for mineral elements absorption was identified in modern wheat cultivars, providing the reference for Zn application in wheat. Meanwhile, this study provides a robust method for quantifying the absorption of mineral elements, which may be adopted into the broadly investigations on the coordinated nutrients absorption of plant.