不同生态型拟南芥耐铵毒害差异的生理机制

Physiological mechanisms of tolerance to ammonium toxicity in different ecotypes of Arabidopsis thaliana

【目的】利用拟南芥生态型群体研究拟南芥耐铵毒害的生理机制,为挖掘耐铵基因提供生理基础及理论指导。【方法】共收集了95份生态型拟南芥材料,采用水培实验方法,将拟南芥幼苗移栽后在正常培养液(2mmol/L NO3–-N处理)中培养8天,然后转移至含有1 mmol/L (NH4)2SO4的营养液(2 mmol/L NH4+-N处理)中培养8天,收获后,测定植株全氮量、地上部游离铵含量,以及谷氨酰胺合成酶(GS)活性;培养3天后取样,采用RT-PCR技术分析根部主要的铵态氮转运蛋白基因AMT1;1和AMT1;2的表达水平;拟南芥幼苗移栽后在正常培养液中培养8天,转移至丰度为5%的1 mmol/L (15NH4)2SO4中培养,分别处理3 h、6 h和24 h取样,用于同位素分析。【结果】2 mmol/L铵态氮处理下拟南芥群体地上部的生长被显著抑制,并且大量游离铵离子累积于地上部,铵态氮下拟南芥群体体内铵含量是对照硝态氮下的1.5倍以上,其中Si-0生态型在铵态氮下铵含量为19.17μmol/g, FW,是对照的20倍。在硝态氮培养条件下,内源铵的含量与拟南芥地上部生长呈显著负相关,铵态氮培养条件下,地上部生长与铵含量同样呈较高的负相关性,因此内源铵含量少的生态型拟南芥在铵态氮下亦耐铵,所以本研究以拟南芥群体组织内铵含量为主因子,筛选出耐铵拟南芥生态型Or-1、Ta-0,HSM和铵敏感拟南芥生态型Rak-2、Lpv-18、Hi-0,结果表明铵敏感生态型在硝态氮下铵含量是耐铵生态型的1.7倍至10倍。耐铵拟南芥生态型铵转运蛋白基因AMT1;1和AMT1;2的表达水平较铵敏感拟南芥高,植株全氮和地上部15N标记试验结果表明,耐铵拟南芥铵态氮吸收速率高于敏感型。并且耐铵拟南芥生态型在两种氮形态下其谷氨酰胺合成酶(GS)活性均显著高于铵敏感生态型,在硝态氮培养条件下GS活性是铵敏感生态型的1.1~1.8倍,在铵态氮培养条件下是1.2~1.6倍,说明耐铵拟南芥生态型的铵同化能力强于敏感型。【结论】耐铵生态型拟南芥是通过更高的谷氨酰胺合成酶(GS)活性将大量的游离铵同化以减少植株体内游离铵含量,从而减轻植株铵毒害;而不是通过减少铵态氮的吸收。

【Objectives】The physiological mechanisms of tolerance to ammonium toxicity were researched in order to study tolerant genes and provide physiological basis and theoretical guidance in different ecotypes of Arabidopsis thaliana.【Methods】A hydroponics experiment was used to culture 95 Arabidopsis materials in normal culture medium (2 mmol/L nitrate treatment) for 8 days after Arabidopsis seedlings transplanted, and then transferred to nutrient solution containing 1 mmol/L (NH4)2SO4 (2 mmol/L ammonium treatment) for 8 days. After harvesting, total nitrogen content, free ammonium concentration and glutamine synthase (GS) activity in shoot were measured. After 3 days of cultivation, the expression levels of major ammonium transporter genes AMT1;1 and AMT1;2 in root by RT-PCR were analyzed. Arabidopsis seedlings were cultured in normal medium for 8 days after transferring to 1 mmol/L (15NH4)2SO4 with 5% abundance, shoot was harvested at 3, 6 and 24 hours for isotope analysis.【Results】2 mmol/L ammonium significantly inhibited the growth of shoot in Arabidopsis thaliana population, and ammonium accumulated in the shoot. The ammonium concentration under ammonium application was more than 1.5 times of that under nitrate, and ecotype Si-0 was 19.17 μmol/g, FW under ammonium, which was 20 times of that of the control. The content of endogenous ammonium was negatively correlated with the growth of Arabidopsis thaliana shoot under 2 mmol/L nitrate and 2 mmol/L ammonium treatments. Therefore, we used ammonium content of tissues in 2 mmol/L ammonium treatments as the main factor, screened ammonium-tolerant Arabidopsis ecotypes Or-1, Ta-0, HSM and ammonium-sensitive Arabidopsis ecotypes Rak-2, Lpv-18, Hi-0. The results showed that ammonium concentration in sensitive ecotypes was 1.7 to 10 times higher than that in tolerant Arabidopsis ecotypes under nitrate. The expression levels of AMT1;1 and AMT1;2 in the root of tolerant ecotypes was higher than those in root of sensitive ecotypes. The results of total nitrogen and 15N tracer analysis showed that the ammonium uptake rate in tolerant ecotypes was higher than that of sensitive ecotypes. Moreover, the results of glutamine synthetase (GS) showed that the GS activity of tolerant ecotypes was 1.1 to 1.8 and 1.2 to1.6 times of that of sensitive ecotypes under nitrate cultivation and ammonium cultivation respectively, which indicated the ammonium assimilation capacity of tolerant ecotypes was stronger than that of sensitive Arabidopsis ecotypes.【Conclusions】Ammonium-tolerant Arabidopsis thaliana has higher GS activity to alleviate ammonium toxicity by assimilating large amounts of free ammonium in plants, rather than by reducing ammonium uptake.