NaHCO_3胁迫对烟草的毒害机理研究
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摘要
土壤盐碱化导致作物大量减产,甚至产生不毛之地,在过去的50年里,大量研究已逐渐揭露了盐的毒害机理,但此毒害作用多以中性NaCl为基础,近几年已研究表明碱性NaHCO3和Na2CO3能够产生更大的危害,同时也有研究者认为碱性碳酸盐和中性钠盐对植物是两种不同的胁迫,所以进一步研究碱性盐的毒害机理将有助理解碱性盐加强的毒害作用。本文以烟草(Nicotiαnαtαbαcum Linn.)为研究对象,利用非损伤离子探针技术、激光共聚焦显微镜技术、透射电子显微镜等技术,从Na+的吸收、积累和毒害出发,试图揭示NaHCO3相对于中性NaCl能够对植物产生更严重危害的机理和NaHCO3胁迫相对于NaCl胁迫的不同。主要结论如下：
(1)Na+的吸收和区室化：当NaCl和NaHCO3分别加入到植物生长液中时,根细胞H+的外流速度分别为2.41 pmol cm-2s-1和5.42 pmol cm-2s-1,碱性NaHCO3应激显著增加了H+的外流速度,同时细胞内液泡pH值从5.34变化到6.64,液泡明显碱化。NaHCO3胁迫3天后Na+外排速率增加,但这种外排不受矾酸钠的抑制,同时外排的速率和溶液的pH成一定的正相关性。这些结果表明NaHCO3加入后H+大量外流,液泡碱化,导致细胞膜透性增加,NaHCO3胁迫导致增加的Na+外流速度不是因为Na+/H+反向转运体活性增强,而是因为细胞质中积累高的Na+浓度,细胞透性的增加可能导致细胞选择性吸收功破坏,最终引起离子区室化的失败。
(2)Na+的积累和毒害部位：一个月的盐碱胁迫结果显示NaHCO3胁迫中根冠比例显著降低,叶的蒸腾速率和CO2吸收速率显著降低,生物量积累较同浓度NaCl胁迫显著下降,但NaHCO3胁迫老叶中Na的积累为1.11明显低于同浓度NaCl胁迫下Na积累率1.22,这些结果说明低的根冠比例和蒸腾速率抑制Na的向上运输和积累,Na的危害主要集中于根部,有别于NaCl胁迫中Na积累和危害的主要部位为叶的结论。
(3)根细胞壁代谢与抗氧化酶活性：不同盐碱溶液黑暗处理48h,烟草根细胞壁成分中,可溶性糖和处理液pH成负相关；当处理液pH大于8.5时,溶液pH值和半纤维素HC1和HC2成正相关,表明碱加速糖的分解和流出,同时在高pH下,果胶百分含量降低导致半纤维素百分含量上升,由于在高pH值下24小时内根细胞活力已经丧失,所以果胶含量的下降主要来自于碱对果胶的水解作用。24小时的高浓度盐(400mM NaCl)和碱(200mM NaHCO3,100mM Na2CO3)胁迫总体表现为对根抗氧化酶活性影响大于对叶抗氧化酶活性的影响,并且盐和碱对不同的抗氧化酶存在不同的影响,盐对CAT和GR活力破坏更重,而高pH值对SCD、APX和POD活力存在更大的影响。
(4)细胞器危害与程序化死亡：烟草原生质体胁迫8小时表明NaHCO3胁迫比NaCl胁迫更易造成原生质体的破碎和线粒体的空泡化,透射电镜进一步分析表明在质膜破碎前,细胞内的内膜系统遭到破坏,细胞内出现大面积的裂解区域,同时线粒体空泡化或无线粒体脊。在CaCl2、NaCl胁迫和NaHCO3胁迫下都存在原生质体凋亡的过程,其中NaCl胁迫和NaHCO3胁迫下细胞凋亡率较高,最高分别达到7.57%和10.34%,但是碱性增加后,细胞破碎完全取代凋亡,在100 mmol·L-1 NaHCO3和100mmol·L-1 Na2CO3混合溶液中,基本上很难发现凋亡的存在。到碱性程度更高的Na2CO3溶液,原生质体大部分破碎,说明此时碱性盐对于质膜的溶解占主导地位。
(5)ROS反应及外源乙烯的缓解作用：碱性NaHCO3胁迫导致烟草根尖伸长生长受到抑制,同时根毛长度和新生根毛数量大幅度减少,但是ROS反应并不明显。矾酸钠和ACC虽然也抑制根的伸长生长,但能促进根毛的发育,降低根尖ROS反应。矾酸钠和ACC都能缓解NaHCO3对烟草幼苗的危害,其中矾酸钠有效时间较短,并有毒副作用,而ACC有效时间相对较长。二者的共同点是都能抑制质膜ATP酶的活性,进而抑制植物细胞主动吸收溶液中Na+离子的能力；同时,抑制质膜ATP酶活性也会降低H+泵出细胞外的能力,虽然抑制根的酸性生长,但在碱性溶液环境中避免了细胞质的进一步碱化,进而表明外源乙烯通过调控质膜ATP酶活性缓解NaHCO3进入细胞的速度。
The salinization and alkalization of soil severely reduces agricultural yields and productivity, and even barren. Over the past 50 years, extensive research has gradually revealed the toxic mechanisms of salt, but this toxic mechanism is based mostly on neutral NaCl stress. In recent years some studies have examined that alkaline salts (NaHCO3, Na2CO3) can induce much stronger destructive effects on plants than neutral salts (NaCl), but also some researchers have began to realize that the alkaline salt stress and neutral salt stress are two distinct kinds of stresses for plants, therefore, further study of the toxic mechanism of alkaline salt will help us to understand enhancing toxicity of alkaline salt. In this paper, using the Scanning Ion-selective Electrode Technique (SIET), Laser Scanning Confocal Microscopy Technique and Transmission Electron Microscopy Technique, the toxic mechanism of NaHCO3 was investigated with tobacco (Nicotiana tabacum Linn.) in order to reveal the reasons that NaHCO3 stress can produce more serious harm to plants than NaCl stress and differences from NaHCO3 stress and NaCl stress. The main conclusions were as follows:
(1) Na+ absorption and compartmentation:When NaCl and NaHCO3 were added to the plant growth medium, NaHCO3 shock led to a significant increase in the net H+ efflux (5.42 pmol cm-2s-1 compare to NaCl shock (2.41 pmol cm-2s-1), at the same time, pH value of the intracellular vacuole increased from 5.34 to 6.64, the vacuole was alkalized. The seedlings were treated 3 day by the various salts and alkaline solutions, NaHCO3 stress induced increased Na+ efflux than NaCl stress, but this efflux was not inhibited by sodium vanadate and Na+ efflux was a positive correlation with pH of solution. These results indicated that NaHCO3 adding induced H+ extrusion and vacuole alkalization, which leaded to increase membrane permeability. Increased Na+ efflux was not due to the increase of activity of PM Na+/H+ antiporter, but because of the high sodium concentration in the cytoplasm. It was possible that NaHCO3 adding destructed ion selective absorption and leaded to failure of ion compartmentation.
(2) Na+ accumulation and parts of toxicity:The various salt stress from one month showed the root/shoot ratio, the transpiration rate, the CO2 absorption rates and the biomass were significantly decreased under 100mM NaHCO3 stress than 100mM NaCl stress, but Na accumulated ratio of old leaves was 1.11 significantly lower in the NaHCO3 Stress than Na accumulated rate of 1.22 under the NaCl Stress. These results suggested that lower root/shoot ratio and transpiration rate inhibited the upward transport of water and accumulation of Na. Parts of toxicity was the root under NaHCO3 stress, it is different Parts of toxicity was old leaves under NaCl stress.
(3) Cell wall metabolism and antioxidant enzyme activity:The seedlings were treated 48h by various saline and alkaline solutions in dark, soluble sugar of the root was negative correlated with pH of solution. When pH of solution was greater than 8.5, HC1 and HC2 content of cellulose was positively correlated with pH value of solution. These results indicated that alkali accelerated the decomposition of soluble sugar and out of cell. At high pH, increased hemicellulose percentage was due to decreased pectin percentage. Because of cell viability of the root has been lost within 24h, so the decrease in pectin content was mainly from the alkali hydrolysis of pectin. The antioxidant enzyme activities in the roots were greater impacted than the antioxidant enzyme activities in the leaves when tobaccos were treated 24h by salt (400mM NaCl) of and alkali (200mM NaHCO3, 100mM Na2CO3) stress. The different salt and alkali affected on activity of different antioxidant enzymes. With increased Na+ concentration, CAT and GR activity decreased more rapid; SOD, APX and POD activity were greater inhibited with pH changes.
(4) Organelle damage and programmed cell death:The protoplasts of tobacco were treated by various salt and alkali solution, NaHCO3 stress caused more broken and vacuolization of mitochondria, transmission electron microscopy analysis showed that endomembrane system has been damaged before the plasma membrane were damaged, the cytoplasm had lysed regions, mitochondria with dilated or absent cristae. The programmed death of protoplast occurred in all treatment, the high apoptosis rate been found in 200mM NaHCO3 stress and 200mM NaCl stress, it was respectively 10.34% and 7.57%. With the pH increased, Cell apoptosis was completely replaced by broken. These results indicated that alkaline salt could dissolute plasma membrane in high pH environment.
(5) ROS response and function of exogenous ethylene:NaHCO3 stress inhibited root elongation, root hair length of tobacco and also significantly reduced the number of root hairs, but the ROS response was not obvious. Although sodium vanadate and ACC also inhibited root elongation, but can promote the development of root hairs, reduced apical ROS response. Lower NaHCO3 stress was weakened by Sodium vanadate and ACC, but sodium vanadate was effective in the short time and ACC was effective in the relatively long time. Sodium vanadate and ACC could inhibited PM ATPase activity and also inhibited absorption of Na+. ACC reduced H+ efflux to avoid further cytoplasmic alkalinization in NaHCO3 solution although they inhibited the root growth.
(1)Na+的吸收和区室化：当NaCl和NaHCO3分别加入到植物生长液中时,根细胞H+的外流速度分别为2.41 pmol cm-2s-1和5.42 pmol cm-2s-1,碱性NaHCO3应激显著增加了H+的外流速度,同时细胞内液泡pH值从5.34变化到6.64,液泡明显碱化。NaHCO3胁迫3天后Na+外排速率增加,但这种外排不受矾酸钠的抑制,同时外排的速率和溶液的pH成一定的正相关性。这些结果表明NaHCO3加入后H+大量外流,液泡碱化,导致细胞膜透性增加,NaHCO3胁迫导致增加的Na+外流速度不是因为Na+/H+反向转运体活性增强,而是因为细胞质中积累高的Na+浓度,细胞透性的增加可能导致细胞选择性吸收功破坏,最终引起离子区室化的失败。
(2)Na+的积累和毒害部位：一个月的盐碱胁迫结果显示NaHCO3胁迫中根冠比例显著降低,叶的蒸腾速率和CO2吸收速率显著降低,生物量积累较同浓度NaCl胁迫显著下降,但NaHCO3胁迫老叶中Na的积累为1.11明显低于同浓度NaCl胁迫下Na积累率1.22,这些结果说明低的根冠比例和蒸腾速率抑制Na的向上运输和积累,Na的危害主要集中于根部,有别于NaCl胁迫中Na积累和危害的主要部位为叶的结论。
(3)根细胞壁代谢与抗氧化酶活性：不同盐碱溶液黑暗处理48h,烟草根细胞壁成分中,可溶性糖和处理液pH成负相关；当处理液pH大于8.5时,溶液pH值和半纤维素HC1和HC2成正相关,表明碱加速糖的分解和流出,同时在高pH下,果胶百分含量降低导致半纤维素百分含量上升,由于在高pH值下24小时内根细胞活力已经丧失,所以果胶含量的下降主要来自于碱对果胶的水解作用。24小时的高浓度盐(400mM NaCl)和碱(200mM NaHCO3,100mM Na2CO3)胁迫总体表现为对根抗氧化酶活性影响大于对叶抗氧化酶活性的影响,并且盐和碱对不同的抗氧化酶存在不同的影响,盐对CAT和GR活力破坏更重,而高pH值对SCD、APX和POD活力存在更大的影响。
(4)细胞器危害与程序化死亡：烟草原生质体胁迫8小时表明NaHCO3胁迫比NaCl胁迫更易造成原生质体的破碎和线粒体的空泡化,透射电镜进一步分析表明在质膜破碎前,细胞内的内膜系统遭到破坏,细胞内出现大面积的裂解区域,同时线粒体空泡化或无线粒体脊。在CaCl2、NaCl胁迫和NaHCO3胁迫下都存在原生质体凋亡的过程,其中NaCl胁迫和NaHCO3胁迫下细胞凋亡率较高,最高分别达到7.57%和10.34%,但是碱性增加后,细胞破碎完全取代凋亡,在100 mmol·L-1 NaHCO3和100mmol·L-1 Na2CO3混合溶液中,基本上很难发现凋亡的存在。到碱性程度更高的Na2CO3溶液,原生质体大部分破碎,说明此时碱性盐对于质膜的溶解占主导地位。
(5)ROS反应及外源乙烯的缓解作用：碱性NaHCO3胁迫导致烟草根尖伸长生长受到抑制,同时根毛长度和新生根毛数量大幅度减少,但是ROS反应并不明显。矾酸钠和ACC虽然也抑制根的伸长生长,但能促进根毛的发育,降低根尖ROS反应。矾酸钠和ACC都能缓解NaHCO3对烟草幼苗的危害,其中矾酸钠有效时间较短,并有毒副作用,而ACC有效时间相对较长。二者的共同点是都能抑制质膜ATP酶的活性,进而抑制植物细胞主动吸收溶液中Na+离子的能力；同时,抑制质膜ATP酶活性也会降低H+泵出细胞外的能力,虽然抑制根的酸性生长,但在碱性溶液环境中避免了细胞质的进一步碱化,进而表明外源乙烯通过调控质膜ATP酶活性缓解NaHCO3进入细胞的速度。
The salinization and alkalization of soil severely reduces agricultural yields and productivity, and even barren. Over the past 50 years, extensive research has gradually revealed the toxic mechanisms of salt, but this toxic mechanism is based mostly on neutral NaCl stress. In recent years some studies have examined that alkaline salts (NaHCO3, Na2CO3) can induce much stronger destructive effects on plants than neutral salts (NaCl), but also some researchers have began to realize that the alkaline salt stress and neutral salt stress are two distinct kinds of stresses for plants, therefore, further study of the toxic mechanism of alkaline salt will help us to understand enhancing toxicity of alkaline salt. In this paper, using the Scanning Ion-selective Electrode Technique (SIET), Laser Scanning Confocal Microscopy Technique and Transmission Electron Microscopy Technique, the toxic mechanism of NaHCO3 was investigated with tobacco (Nicotiana tabacum Linn.) in order to reveal the reasons that NaHCO3 stress can produce more serious harm to plants than NaCl stress and differences from NaHCO3 stress and NaCl stress. The main conclusions were as follows:
(1) Na+ absorption and compartmentation:When NaCl and NaHCO3 were added to the plant growth medium, NaHCO3 shock led to a significant increase in the net H+ efflux (5.42 pmol cm-2s-1 compare to NaCl shock (2.41 pmol cm-2s-1), at the same time, pH value of the intracellular vacuole increased from 5.34 to 6.64, the vacuole was alkalized. The seedlings were treated 3 day by the various salts and alkaline solutions, NaHCO3 stress induced increased Na+ efflux than NaCl stress, but this efflux was not inhibited by sodium vanadate and Na+ efflux was a positive correlation with pH of solution. These results indicated that NaHCO3 adding induced H+ extrusion and vacuole alkalization, which leaded to increase membrane permeability. Increased Na+ efflux was not due to the increase of activity of PM Na+/H+ antiporter, but because of the high sodium concentration in the cytoplasm. It was possible that NaHCO3 adding destructed ion selective absorption and leaded to failure of ion compartmentation.
(2) Na+ accumulation and parts of toxicity:The various salt stress from one month showed the root/shoot ratio, the transpiration rate, the CO2 absorption rates and the biomass were significantly decreased under 100mM NaHCO3 stress than 100mM NaCl stress, but Na accumulated ratio of old leaves was 1.11 significantly lower in the NaHCO3 Stress than Na accumulated rate of 1.22 under the NaCl Stress. These results suggested that lower root/shoot ratio and transpiration rate inhibited the upward transport of water and accumulation of Na. Parts of toxicity was the root under NaHCO3 stress, it is different Parts of toxicity was old leaves under NaCl stress.
(3) Cell wall metabolism and antioxidant enzyme activity:The seedlings were treated 48h by various saline and alkaline solutions in dark, soluble sugar of the root was negative correlated with pH of solution. When pH of solution was greater than 8.5, HC1 and HC2 content of cellulose was positively correlated with pH value of solution. These results indicated that alkali accelerated the decomposition of soluble sugar and out of cell. At high pH, increased hemicellulose percentage was due to decreased pectin percentage. Because of cell viability of the root has been lost within 24h, so the decrease in pectin content was mainly from the alkali hydrolysis of pectin. The antioxidant enzyme activities in the roots were greater impacted than the antioxidant enzyme activities in the leaves when tobaccos were treated 24h by salt (400mM NaCl) of and alkali (200mM NaHCO3, 100mM Na2CO3) stress. The different salt and alkali affected on activity of different antioxidant enzymes. With increased Na+ concentration, CAT and GR activity decreased more rapid; SOD, APX and POD activity were greater inhibited with pH changes.
(4) Organelle damage and programmed cell death:The protoplasts of tobacco were treated by various salt and alkali solution, NaHCO3 stress caused more broken and vacuolization of mitochondria, transmission electron microscopy analysis showed that endomembrane system has been damaged before the plasma membrane were damaged, the cytoplasm had lysed regions, mitochondria with dilated or absent cristae. The programmed death of protoplast occurred in all treatment, the high apoptosis rate been found in 200mM NaHCO3 stress and 200mM NaCl stress, it was respectively 10.34% and 7.57%. With the pH increased, Cell apoptosis was completely replaced by broken. These results indicated that alkaline salt could dissolute plasma membrane in high pH environment.
(5) ROS response and function of exogenous ethylene:NaHCO3 stress inhibited root elongation, root hair length of tobacco and also significantly reduced the number of root hairs, but the ROS response was not obvious. Although sodium vanadate and ACC also inhibited root elongation, but can promote the development of root hairs, reduced apical ROS response. Lower NaHCO3 stress was weakened by Sodium vanadate and ACC, but sodium vanadate was effective in the short time and ACC was effective in the relatively long time. Sodium vanadate and ACC could inhibited PM ATPase activity and also inhibited absorption of Na+. ACC reduced H+ efflux to avoid further cytoplasmic alkalinization in NaHCO3 solution although they inhibited the root growth.
引文
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