The Mechanical of Organic Acids Secreted by Roots of Tartary Buckwheat under the Effects of Low Nitrogen Stress

A pot experiment was conducted to study the effects of two different low nitrogen tolerant tartary buckwheat varieties’ (Diqing buckwheat (DQ, low nitrogen resistance) and Heifeng 1 (HF, low nitrogen sensitive) response mechanism of organic acids to low nitrogen stress. The results showed that the soil moisture of HF and DQ under low nitrogen treatment decreased 24.2% and 14.32%, respectively when compared with normal nitrogen treatment, and the water consumption of DQ was significantly higher than that of HF at seedling stage. Under low nitrogen treatment, the soil pH value of DQ was 1.44% and 8.44% lower than that of HF at seedling and flowering stages, respectively, the content of NH4+ in DQ soil was 8.2% lower than that of HF at maturity stage, the content of NO3− was significantly higher than that HF 49.2%, 12.9%, and 16.6% in each growth period, respectively. Split plot analysis showed that nitrogen treatment significantly affected the organic acids content in the soil of the buckwheat. The secretion content of organic acids are different among buckwheat cultivars under low nitrogen stress. In the soil of DQ, the content of malonic acid was higher than that of HF by 34.39% at maturity stage; the content of oxalic acid was respectively higher than that of HF by 24.86% and 24.52% at seedling and flowering stages; the content of propionic acid was significantly higher than that of HF by 7.36%, 9.44% and23.47% in each growth period, respectively; and tartaric acid acetic acid also showed the same trend at flowering and maturity stages. In summary, tartary buckwheat may regulate the nutrient availability of rhizosphere soil through the secretion of organic acids in the root system to cope with the low nitrogen stress environment. For the cultivation of tartary buckwheat on poor soil should consider the differences cultivaring barren resistance varieties to increase efficiency in the future.


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Nitrogen is one of the basic nutrients required for plant growth, which affects crop 34 growth、 yield and quality [1]. The lack of nitrogen can lead to slow plant growth, 35 decrease yields, and reduce quality. Therefore, the soil must contain enough nitrogen to 36 meet the normal growth of plants [2]. But nitrogen is a limiting factor in many arable 37 soils in China. For example, the major characteristics of Loess Plateau soils are nitrogen 38 deficiency, low phosphorus and sufficient potassium so that a large amount of nitrogen 39 fertilizer is applied to ensure yield during planting [3]. However, nitrogen fertilizers have 40 lower utilization efficiency, the loss of a large amount of nitrogen not only causes huge 41 waste, but also causes problems such as water bodies and environmental pollution [3]. 42 Therefore, focusing on the overall objective of building a green, efficient and sustainable 43 agricultural production system, the selection of crops with low nitrogen tolerance can not 44 only ensure the normal growth of crops, but also control the amount of nitrogen fertilizer 45 and improve the utilization efficiency of nitrogen and it has significance for improving 46 the ecological environment [4]. 47 Many scholars have conducted research on the low-nitrogen-resistant characteristics 48 of large crops such as wheat and corn [5], but there are few researches on small grains 49 such as tartary buckwheat. Most of the buckwheat is grown in the alpine region, which 50 has the characteristics of short fertility cycle, strong adaptability, drought tolerance and but few studies on the underground parts of tartary buckwheat, especially the response 55 mechanism of tartary buckwheat to low-nitrogen stress are rarely reported. 56 Studies have shown that after suffering stress from soil nutrient, plant roots first feel 57 the stress and react quickly to adapt to the stress environment through a series changes of 58 root physiological, especially some stress conditions can induce the plant roots to secrete 59 large amounts of organic acids, which is a common active adaptive response [8]. The 60 articles on organic acids secreted by root system under ,low nitrogen stress mainly focus 61 on oats, rice and other crops, and also have different results. With the growth of oats, the 62 total amount of organic acids gradually decreased in root exudates, and the total amount 63 of organic acids secreted by the oat roots under nitrogen stress was significantly higher 64 than that of nitrogen supply [ (low nitrogen treatment), N2 (normal nitrogen treatment), and were applied to the same 92 amount of phosphate and potassium fertilizer. Urea (46.4% of nitrogen content) was 93 applied of 80 mg kg -1 to low nitrogen treatment and 160 mg kg -1 to normal nitrogen 94 treatment, phosphate fertilizer was P 2 O 5 with 150 mg kg -1 , and potassium fertilizer was     (Table 1). In 134 the seedling stage, the soil moisture of HF and DQ under Low N treatment decreased by 135 24.2% and 14.32%, respectively when compared with the normal N treatment. In the 136 flowering stage, the water consumption of HF under low N treatment was higher than that 137 of normal N treatment, while DQ had no significant difference. In the maturity stage, HF 138 had no significant difference, however the water consumption of DQ is larger than normal 139 N treatment. Under low N treatment, the soil water content of HF was 33.64% higher than 140 DQ in seedling stage, but no significant difference in flowering and maturity (Table 1).

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This indicates that the water consumption of DQ is significantly higher than that of HF 142 under the low N treatment at seedling stage.  (Table 1). At maturity, there was no significant difference in 148 DQ, however the soil pH of HF was 1.25% higher than that of normal N treatment. The 149 effects of different cultivars on soil pH mainly concentrated in the seedling stage and the 150 flowering stage, the soil pH value of DQ was lower than that of HF by 1.44% and 8.44%

Effect of buckwheat cultivars and nitrogen treatments on
There was no significant difference in NH 4 + content of HF soil between N1 and N2 155 at different growth stages, but they all showed significant differences with CK. They were 156 1.192 times and 1.373 times higher than CK at seedling stage , and 17.8% and 27.5% 157 higher than CK at flowering stage , and 80.6% and 68% higher than CK at maturity stage, 158 respectively ( Table 1). The content of NH 4 + in DQ soil showed significant differences 159 under different nitrogen treatments during growth period, N1 was lower than N2 of 160 16.2% and 8.39% at seedling and flowering stage, respectively, and N1 was higher than 161 N2 of 13.72% at maturity stage (Table 1). Under low N treatment, the content of NH 4 + in 162 DQ soil had no significant difference with HF at seedling and flowering stage, but was 163 lower than that of HF 8.2% at maturity stage (Table 1).

Effect of buckwheat cultivars and nitrogen treatments on
Under N1 treatment, the content of NO 3 − in HF soil was lower than N2 of 30.7% , 167 but higher than N2 of 14.3% in DQ soil at seedling stage. the content of NO 3 − in HF and 168 DQ soils were higher than N2 10.6% and 16% respectively at flowering stage, and were 169 lower than N2 23.8% and 8.8% respectively at maturity stage (Table 1). Under low N 170 treatment, the content of NO 3 − in DQ soil was significantly higher than that of HF 49.2%, 171 12.9%, and 16.6% in each growth period, respectively (Table 1). that of HF at seedling and flowering stages, but higher than that of HF by 34.39% at 206 maturity stage; The content of oxalic acid was respectively higher than that of HF by 207 24.86% and 24.52% at seedling and flowering stages, but lower than that of HF by 208 24.65% at maturity stage; the content of propionic acid was significantly higher than that 209 of HF by 7.36%, 9.44% and 23.47% in each growth period; tartaric acid and acetic acid 210 also showed the same trend, their contents were higher than that of HF by 56.91% and 211 59.6% at flowering stage, and higher than that of HF by 24.97% and 18.51% at maturity 212 stage, respectively (Fig 3).  In the seedling stage (S), the first axis explained 95.9% of variation ,which clearly 218 separate N2 from CK, and the second axis clearly separated the two varieties (HF and 219 DQ). In the flowering stage (F), the first axis explained 96.1% of variation, which clearly 220 separated N2 from the other N treatments (CK, N1), but the difference between the two 221 varieties was not significant. In the maturity stage (M), the first axis explained 99.2% of 222 the variation, which clearly separated the two varieties under N1 treatment, but the 223 difference between two varieties was not significant under other N treatments, and the 224 second axis clearly separates CK from the other N treatments (N1, N2) (Fig 4). Except for 225 oxalic acid, the contents of the other four organic acids in the soil showed significant 226 positive correlations at different growth periods, and were negatively correlated with soil 227 moisture and pH, and positively correlated with NH 4 + andNO 3 - (Fig 4).  summary, a large number of organic acids may be secreted by the roots of buckwheat to 296 adapt to low N stress. At the same time, these organic acids will promote the conversion 297 of NH 4 + to NO 3 − , which will increase the content of NO 3 − in the soil and accelerate the 298 absorption of nitrogen. Whether this judgment is correct or not requires further study.

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In this experiment, the contents of several organic acids detected in the soils of two 300 buckwheat cultivars were lower than that of normal N treatment, the content of NH 4 + 301 detected in the DQ soil was higher than that of the normal N treatment, and the content of 302 NO 3 − detected in the two buckwheat soils was lower than that of the normal N treatment 303 under low N treatment at maturity stage, which may be due to that plant life activity is 304 basically completed at maturity, the demand for nutrients is reduced, and the content of 305 various organic acids secreted by the root system is also decreased, simultaneously the 306 nitrogen content in the soil was less at maturity. Therefore, the content of NO 3 − that can 307 be absorbed was less under low N treatment, and then the contents of organic acids 308 secreted by the root system also decreased. At the same time, the promotion of the 309 conversion of NH 4 + to NO 3 − was reduced, resulting in more NH4+ content and less NO 3 − 310 content in the soil .Low N or N deficiency accelerated the root senescence, the root 311 exudation ability was significantly decreased, and the organic acids secreted by the root 312 decreased in varying degrees [10]. In the above study, the pH of DQ soil was higher than 313 that of normal N treatment at seedling stage and flowering stage and the pH of HF soil 314 was higher than that of normal N treatment at maturity under low N treatment. This may nitrogen treatment, the content of NH 4 + in DQ soil was lower than that of HF at maturity, 359 and the content of NO 3 − in DQ soil was higher than that of HF at different growth periods.

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This may be due to that low-nitrogen-tolerant cultivars have a stronger ability to 361 transform nitrogen under low N stress, and convert it from urea to NH 4 + , then they can be 362 rapidly transformed into NO 3 − which is better absorbed by the plants, thus the soils of 363 low-nitrogen-tolerant cultivars have a high content of NO 3 − . However, the content of 364 NH 4 + in low-nitrogen-tolerant soils was lower at maturity stage, which may be due to the 365 large amount of NH 4 + converted to NO 3 − [33]. In addition, the soil pH of DQ was lower 366 than that of HF at seedling and flowering stage under low N treatment, probably because 367 DQ secreted more organic acids. It turns out that the contents of several organic acids in 368 DQ soil were higher than those in HF under low N stress. In summary, barren-resistant 369 cultivars may adapt to low N stress by secreting more organic acids. Under low N stress,