孙黎明(1990—),男,湖北仙桃人,博士研究生,主要从事水稻逆境生理研究。Email:
酸毒是酸性土壤中限制作物生长的重要因子之一,但酸毒通常与金属离子毒性共存,难以在土壤中直接研究,目前关于水稻酸毒机制的报道较少。选用前期筛选的酸耐性不同的两个水稻品种Kasalath(酸耐性)和Jinguoyin(酸敏感),研究水稻的酸敏感性与活性氧(ROS)积累及氧化还原代谢相关酶的关系,并试图探讨酸毒害中一氧化氮(NO)信号与活性氧信号的调控关系。结果显示,低pH引起酸敏感水稻品种Jinguoyin中根尖NO和ROS的富集,但酸耐性水稻品种Kasalath中无显著变化。NO清除剂2-(4-羧基苯基)-4, 4, 5, 5-四甲基咪唑啉-1-氧基-3-氧化物钾盐(cPTIO)可清除Jinguoyin根尖富集的NO和ROS。硝酸还原酶反馈抑制剂谷氨酰胺(Gln)可明显降低Jinguoyin在低pH下的根尖NO信号,而一氧化氮合酶抑制剂N'-硝基-L-精氨酸甲酯盐酸盐(L-NAME)对根尖NO信号无影响。低pH显著提高了Jinguoyin中硝酸还原酶基因
Low pH stress is one of the major factors limiting crop production on acidic soils. It often coexists with metal ion toxicity and this makes it difficult to explore directly the effect of pH in acidic soils. So far, the mechanisms of low pH stress in rice is poorly understood.
Two rice varieties with different low pH tolerance, Kasalath (low pH-tolerant) and Jinguoyin (low pH-sensitive), were selected to (i) study the relationship between low pH stress and the accumulation of nitric oxide (NO) and reactive oxygen species (ROS), and (ii) explore the regulatory relationship between NO and ROS under low pH stress.
Low pH caused the accumulation of NO and ROS in the root tips of Jinguoyin, but there was no significant change in Kasalath. The NO scavenger cPTIO reduced NO and ROS accumulation in root tips of Jinguoyin. Feedback inhibitor of nitrate reductase Gln significantly reduced NO content in the root tips of Jinguoyin under low pH, while L-NAME, a nitric oxide synthase inhibitor, did not affect NO content in the root tips of Jinguoyin. Low pH significantly increased the expression of nitrate reductase genes
Low pH stress of Jinguoyin was related to the NO-mediated ROS accumulation. The NO signal generated under low pH stress is mainly synthesized by nitrate reductase through increasing the expression of
酸性土壤(pH < 5.5)约占世界耕地面积的40%、潜在可耕作土地的70%[
低pH下,土壤中的氢离子浓度较高,常常引起土壤中金属离子如铝、铁和锰等的活化而间接造成植物伤害[
一氧化氮(NO)是植物体中的一种重要气体信号分子,在植物生长发育和逆境响应中发挥重要作用[
本研究选用酸耐性不同的两个籼稻品种Kasalath(酸耐性)和Jinguoyin(酸敏感)。水稻种子用10% H2O2消毒10 min,用去离子水洗净后在30℃恒温箱中避光浸泡催芽2 d。随后将刚露白的水稻种子转移至0.5 mmmol·L–1 CaCl2(pH 5.6)溶液的浮板上,26℃暗培养,CaCl2溶液每两天更换一次。
选择生长约5 d、整齐一致的水稻幼苗,分别在pH为4.2、4.5和4.8的含有0.5 mmmol·L–1 CaCl2水溶液中处理24 h,每个处理10株。用直尺测定处理前后根长,相减即为根伸长。
根尖细胞膜的完整性采用伊文思蓝(Evans blue)吸收量的方法[
抗氧化酶包括抗坏血酸过氧化物酶(APX,BC0220)、过氧化氢酶(CAT,BC0200)、过氧化物酶(POD,BC0090)和超氧化物歧化酶(SOD,BC0170)的活性均采用索莱宝的试剂盒进行测定。还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性的测定采用植物NADPH氧化酶(NADPH-OX)ELISA试剂盒(MB-10702A,酶标生物,江苏)。根尖蛋白质的提取根据Tian等[
根尖ROS的测定采用二氢乙啶(Dihydroethidium,DHE)荧光染料法。将处理24 h后的水稻根尖切下,置于1 mL 10 μmmol·L–1 DHE的水溶液中避光37℃孵育30 min,蒸馏水冲洗3次后,用荧光显微镜(Nikon,Minato,Tokyo,日本)拍照,使用的滤光片为EX 540/25、DM565、BA605/55。根系NO含量的测定采用3-氨基-4-甲氨基-2’-7’-二氟荧光素(DAF-FM DA)进行测定。将水稻根尖1 cm剪下,在20 mmmol·L–1的4-羟乙基哌嗪乙磺酸(HEPES)缓冲液(pH7.4)中清洗20 min,然后将根尖置于含10 μmmol·L–1荧光染料的1.5 mL离心管中避光染色30 min,然后在20 mmmol·L–1的HEPES缓冲液(pH7.4)避光清洗三次,每次15 min,制片,在荧光显微镜(Nikon,Minato,Tokyo,日本)下观察拍片,使用的滤光片为EX 465-495、DM 505和BA 512-558。
每个处理取处理后的15~20条根尖(0~1 cm),利用液氮研磨粉碎,重复4次。RNA的提取使用TGuide Cells/Tissue/Plant RNA Kit(天根,北京)。获得的RNA用Nanodrop 2000C(Thermo Fisher,美国)测定浓度和质量,并在1%琼脂糖上电泳检查完整性。用PrimeScript RT试剂盒(Takara,日本)反转录合成cDNA。使用SYBR Premix ExTaq(Takara,日本)在Roche LightCycler 480 II上进行实时荧光定量PCR。以水稻
文中使用最小显著差异法(LSD)进行方差分析,SigmaPlot 14.0软件进行作图。
培养液pH从4.8降至4.2,酸敏感水稻品种Jinguoyin的根伸长显著降低,根尖出现卷曲,而酸耐性品种Kasalath的根伸长并未受到明显影响(
pH对Kasalath和Jinguoyin根伸长的影响
Effect of pH on the root elongation in Kasalath and Jinguoyin
MDA是膜脂过氧化最重要的产物之一,pH从4.8下降至4.2,Jinguoyin根尖MDA浓度和伊文思蓝吸收量显著升高(
pH对Kasalath和Jinguoyin根尖细胞膜完整性和过氧化的影响
Effect of pH on cell membrane integrity and peroxide in the root tips of Kasalath and Jinguoyin
pH从4.8下降至4.2,Kasalath和Jinguoyin根尖SOD和APX活性均显著增加,且Jinguoyin的增加幅度更大(
pH对Kasalath和Jinguoyin根尖抗氧化酶系统和还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶活性的影响
Effect of pH on the activity of antioxidant enzymes and NADPH oxidase in the root tips of Kasalath and Jinguoyin
培养液pH从4.8下降至4.2,酸敏感品种Jinguoyin根尖NO和ROS信号均明显增强,而酸耐性品种Kasalath根尖NO和ROS信号并未明显变化(
pH对Kasalath和Jinguoyin根尖NO和ROS信号的影响
Effect of pH on NO and ROS in the root tips of Kasalath and Jinguoyin
为了探明酸毒诱导的NO和ROS信号的上下游调控关系,通过清除根尖NO来确定NO对ROS和根伸长的影响。添加NO清除剂2-(4-羧基苯基)- 4, 4, 5, 5-四甲基咪唑啉-1-氧基-3-氧化物钾盐(cPTIO)可以显著提高Jinguoyin在低pH下的根生长(
NO清除剂cPTIO对Jinguoyin根生长(a,b)、根尖NO信号(c)和ROS信号(d)的影响
Effect of NO scavenger cPTIO on root growth(a, b), NO(c)and ROS(d)in the root tips of Jinguoyin
植物中的NO主要由硝酸还原酶和一氧化氮合酶合成,为了确定Jinguoyin中酸毒诱导的NO主要是哪个酶在发挥作用,观察外源添加这两个酶的抑制剂Gln(硝酸还原酶反馈抑制剂)和L-NAME(一氧化氮合成酶抑制剂)对根伸长和NO信号的影响。硝酸还原酶反馈抑制剂Gln可显著提高根系在低pH下的生长,而一氧化氮合酶抑制剂L-NAME未表现出显著的缓解作用(
酶抑制剂Gln和L-NAME对Jinguoyin根生长(a,b)和根尖NO信号(c)的影响
Effect of enzyme inhibitor Gln and L-NAME on root growth(a, b)and NO(c)in the root tips of Jinguoyin
低pH显著提高Kasalath和Jinguoyin根尖硝酸还原酶活性,而Jinguoyin中的增加幅度显著高于Kasalath(210.3% vs 47.5%)(
pH对Kasalath和Jinguoyin根尖硝酸还原酶活性(a)和一氧化氮合成酶活性(b)的影响
Effect of pH on the activity of nitrate reductase(a)and nitric oxide synthase(b)in the root tips of Kasalath and Jinguoyin
水稻基因组含有3个编码硝酸还原酶的基因,
pH对Kasalath和Jinguoyin根尖
Effect of pH on the expression of
低pH降低根系的H+分泌[
在植物体内,NO作为气体信号分子通过激活抗氧化系统而间接清除ROS,亦可直接与ROS反应而清除ROS,因此它被称为次级抗氧化剂[
目前,已知植物NO的生物合成途径至少有硝酸还原酶途径和一氧化氮合酶途径等7种[
本研究利用耐酸性不同的两个籼稻品种Kasalath和Jinguoyin,发现在酸敏感水稻Jinguoyin中低pH引起的酸毒与NO介导的ROS富集相关,酸毒引起的NO信号主要与硝酸还原酶活性的提高有关,而硝酸还原酶活性的提高与硝酸还原酶基因
von Uexküll H R, Mutert E. Global extent, development and economic impact of acid soils[J]. Plant and Soil, 1995, 171(1): 1—15.
Guo J H, Liu X J, Zhang Y, et al. Significant acidification in major Chinese croplands[J]. Science, 2010, 327 (5968): 1008—1010
Food and Agriculture Organization (FAO). World agriculture : Towards 2015/2030 summary report[R]. Rome, 2002.
Iuchi S, Koyama H, Iuchi A, et al. Zinc finger protein STOP1 is critical for proton tolerance in
Kochian L V, Piñeros M A, Liu J P, et al. Plant adaptation to acid soils: The molecular basis for crop aluminum resistance[J]. Annual Review of Plant Biology, 2015, 66: 571—598.
Kobayashi Y, Kobayashi Y, Watanabe T, et al. Molecular and physiological analysis of Al3+ and H+ rhizotoxicities at moderately acidic conditions[J]. Plant Physiology, 2013, 163(1): 180—192.
Wang H H, Li Y, Hou J J, et al. Nitrate reductasemediated nitric oxide production alleviates Al-induced inhibition of root elongation by regulating the ascorbate-glutathione cycle in soybean roots[J]. Plant and Soil, 2017, 410(1/2): 453—465.
Cai M Z, Zhang S N, Wang F M, et al. Protective effect of exogenously applied nitric oxide on aluminum-induced oxidative stress in soybean plants[J]. Russian Journal of Plant Physiology, 2011, 58(5): 791—798.
Sun C L, Lu L L, Liu L J, et al. Nitrate reductasemediated early nitric oxide burst alleviates oxidative damage induced by aluminum through enhancement of antioxidant defenses in roots of wheat (Triticum aestivum)[J]. New Phytologist, 2014, 201 (4): 1240—1250.
Wang H, Huang J, Bi Y. Nitrate reductase-dependent nitric oxide production is involved in aluminum tolerance in red kidney bean roots[J]. Plant Science, 2010, 179(3): 281—288.
Zhang H, Li Y H, Hu L Y, et al. Effects of exogenous nitric oxide donor on antioxidant metabolism in wheat leaves under aluminum stress[J]. Russian Journal of Plant Physiology, 2008, 55(4): 469—474.
Tian Q Y, Sun D H, Zhao M G, et al. Inhibition of nitric oxide synthase (NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos[J]. New Phytologist, 2010, 174(2): 322—331.
Bose J, Babourina O, Shabala S, et al. Aluminumdependent dynamics of ion transport in Arabidopsis: Specificity of low pH and aluminum responses[J]. Physiologia Plantarum, 2010, 139(4): 401—412.
Lager I, Andréasson O, Dunbar T L, et al. Changes in external pH rapidly alter plant gene expression and modulate auxin and elicitor responses[J]. Plant, Cell & Environment, 2010, 33(9): 1513—1528.
Tsang D L, Edmond C, Harrington J L, et al. Cell wall integrity controls root elongation via a general 1-aminocyclopropane-1-carboxylic acid-dependent, ethylene-independent pathway[J]. Plant Physiology, 2011, 156(2): 596—604.
Yan F, Schubert S, Mengel K. Effect of Low Root Medium pH on Net Proton Release, Root Respiration, and Root Growth of Corn(
Monshausen G B, Bibikova T N, Messerli M A, et al. Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(52): 20996—21001.
Tanimoto E, Fujii S, Yamamoto R, et al. Measurement of viscoelastic properties of root cell walls affected by low pH in lateral roots of
Koyama H, Toda T, Hara T. Brief exposure to low-pH stress causes irreversible damage to the growing root in
Chen H F, Zhang Q, Zhang Z H. Comparative transcriptome combined with metabolomic and physiological analyses revealed ROS-mediated redox signaling affecting rice growth and cellular iron homeostasis under varying pH conditions[J]. Plant and Soil, 2019, 434(1/2): 343—361.
Long A, Huang W L, Qi Y P, et al. Low pH effects on reactive oxygen species and methylglyoxal metabolisms in
Yang T Y, Huang W T, Zhang J, et al. Raised pH conferred the ability to maintain a balance between production and detoxification of reactive oxygen species and methylglyoxal in aluminum-toxic
Gill S S, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants[J]. Plant Physiology and Biochemistry, 2010, 48 (12): 909—930.
Chen H F, Zhang Q, Zhang Z H. Comparative transcriptome combined with metabolomic and physiological analyses revealed ROS-mediated redox signaling affecting rice growth and cellular iron homeostasis under varying pH conditions[J]. Plant and Soil, 2019, 434(1/2): 343—361.
Zhang Y Y, Zhang W H, Xue L, et al. Advance on NO function in plant growth, development and abiotic stress tolerance[J]. Acta Botanica Boreali-Occidentalia Sinica, 2012, 32(4): 835—842.
张艳艳, 章文华, 薛丽, 等. 一氧化氮在植物生长发育和抗逆过程中的作用研究进展[J]. 西北植物学报, 2012, 32(4): 835—842.
Xia H W, Shi G X, Huang M, et al. Advances on effects of nitric oxide on resistances of plants to heavy metal stress[J]. Acta Ecologica Sinica, 2015, 35 (10): 3139—3147.
夏海威, 施国新, 黄敏, 等. 一氧化氮对植物重金属胁迫抗性的影响研究进展[J]. 生态学报, 2015, 35(10): 3139—3147.
Xiong J, Fu G F, Yang Y J, et al. Roles of nitric oxide in growth of plant root[J]. Journal of Huazhong Agricultural University, 2011, 30(3): 375—383.
熊杰, 符冠富, 杨永杰, 等. 一氧化氮在植物根系生长发育过程中的作用研究进展[J]. 华中农业大学学报, 2011, 30(3): 375—383.
Gupta K J, Fernie A R, Kaiser W M, et al. On the origins of nitric oxide[J]. Trends in Plant Science, 2011, 16(3): 160—168.
Sahay S, Gupta M. An update on nitric oxide and its benign role in plant responses under metal stress[J]. Nitric Oxide, 2017, 67: 39—52.
Yamamoto Y, Kobayashi Y, Matsumoto H. Lipid Peroxidation Is an Early Symptom Triggered by Aluminum, But Not the Primary Cause of Elongation Inhibition in Pea Roots[J]. Plant Physiology, 2001, 125(1): 199—208.
Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72(1/2): 248–254.
Araki R, Kousaka K, Namba K, et al. 2'‐ Deoxymugineic acid promotes growth of rice(
Hao F, Wang X, Chen J. Involvement of plasmamembrane NADPH oxidase in nickel-induced oxidative stress in roots of wheat seedlings[J]. Plant Science, 2006, 170(1): 151—158.
Scharte J, Schön H, Tjaden Z, et al. Isoenzyme replacement of glucose-6-phosphate dehydrogenase in the cytosol improves stress tolerance in plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(19): 8061—8066.
Gilroy S, Suzuki N, Miller G, et al. A tidal wave of signals: calcium and ROS at the forefront of rapid systemic signaling[J]. Trends in Plant Science, 2014, 19 (10): 623—630.
Gilroy S, Białasek M, Suzuki N, et al. ROS, calcium, and electric signals: Key mediators of rapid systemic signaling in plants[J]. Plant Physiology, 2016, 171(3): 1606—1615.
Choudhury F K, Rivero R M, Blumwald E, et al. Reactive oxygen species, abiotic stress and stress combination[J]. Plant Journal, 2017, 90(5): 856—867.
Cao X C, Zhu C Q, Zhong C, et al. Nitric oxide synthase-mediated early nitric oxide burst alleviates water stress-induced oxidative damage in ammoniumsupplied rice roots[J]. BMC Plant Biology, 2019, 19(1): 108.
Wang H H, Hou J J, Li Y, et al. Nitric oxide-mediated cytosolic glucose-6-phosphate dehydrogenase is involved in aluminum toxicity of soybean under high aluminum concentration[J]. Plant and Soil, 2017, 416(1/2): 39—52.
Kaiser W M, Brendle-Behnisch E. Acid-base-modulation of nitrate reductase in leaf tissues[J]. Planta, 1995, 196(1): 1—6.
Ferrari T E, Varner J E. Intact tissue assay for nitrite reductase in barley aleurone layers[J]. Plant Physiology, 1971, 47(6): 790—794.