【作者】 王玉倩；

【导师】 封克；

【作者基本信息】 扬州大学 ， 植物学， 2010， 博士

【摘要】 钾是植物必需的大量元素之一,在植物体内发挥重要的生理功能。与其理化性质相近的钠却是引起植物盐害的主要离子之一。研究发现,细胞质内较高的钾/钠比对维持植物在盐胁迫环境下的正常生长至关重要,即植物根系对钾、钠的吸收与其耐盐性之间关系密切。前人对于钾吸收的研究主要集中在专性钾载体和专性钾通道上,然而越来越多的证据显示,非选择性阳离子通道（NSCCs）在多种阳离子的吸收方面具有重要的作用。NSCCs可以介导双向离子流,不仅参与K⁺吸收,而且与K⁺流出有关,因此NSCCs可能从两方面调节植物细胞质内的K⁺浓度。NSCCs同时还是Na⁺进入细胞的主要途径。由于NSCCs具有这些钾的专性通道所不具备的独特性质,因此可以推测,NSCCs不仅可以直接影响植物对钾离子的吸收,也可以通过对钾钠离子的双向转运从而调节体内的钾/钠比。但迄今为止,有关NSCCs在植物的钾营养吸收方面所起的作用以及该通道系统的钾转移过程受何种因素的影响等尚未能进行过系统研究。本文所采用的研究思路是首先筛选耐盐和盐敏感的不同小麦品种,然后对其吸钾状况进行对比,在添加专性抑制剂的条件下了解NSCCs在不同品种的吸钾过程中所作的贡献,在此基础上进一步观察哪些因素影响着NSCCs的功能,以及钾在通过NSCCs的转移过程中细胞膜性质所发生的相应变化。希望通过这些研究,能够从NSCCs的角度发现出耐盐能力不同的小麦基因型在营养吸收生理机制和耐盐机制方面的差异,为培育耐盐品种提供参考。试验材料为小麦,分别为耐盐性强的石家庄8号、耐盐中等的扬麦16号和盐敏感的苏徐2号。本文所涉及的主要研究方法为:试验用苗采用水培法,钾离子的吸收速率采用常规动力学方法,细胞膜电位测定采用玻璃微电极法。采用钾载体及钾通道的专性抑制剂的方法将NSCCs的吸钾作用从植物总吸钾作用中分离出来。通过研究得到以下主要结论:（1）小麦耐盐能力与其本身的吸钾能力有关,吸钾能力越强,耐盐能力越大。在低钾条件下,NSCCs介导钾的外流,高钾条件下NSCCs介导钾的内流。（2）环境条件可以对小麦的根细胞NSCCs转移钾的过程产生影响。较高的温度使耐盐小麦NSCCs转移的钾对总吸钾的贡献率下降,对NSCCs转运钾的影响大于对专一性钾通道的影响;较低温度使通过NSCCs转移的钾对总吸钾的贡献率上升,对专一性钾通道活性的抑制比对NSCCs的抑制更强。（3）重金属镉和铜均对耐盐小麦的钾吸收速率产生影响,浓度越大,抑制作用越强。NSCCs转移钾的过程对镉的敏感度相对于专一性钾通道来说要低,而对铜的敏感度相对于专一性钾通道来说要高。说明不同重金属对两类通道蛋白的影响机制不一样。（4）低盐环境并不降低三种小麦对钾离子的吸收速率,甚至还对总吸钾速率具有一定的促进作用;高盐条件对三种小麦的钾离子的吸收速率均有明显抑制作用。三种小麦根系NSCCs转移钾的过程均受到Na⁺的影响,但影响程度不同:耐盐性最强的石家庄8号,在低盐条件下,其NSCCs的钾转移过程受到的影响较小,在高盐条件下,通过NSCCs的钾转移对总吸钾的贡献率不降反升。盐敏感的苏徐2号,其NSCCs钾转移过程受钠的抑制程度最大。耐盐程度中等的扬麦16号,NSCCs受到的影响居中。由此可见,NSCCs的钾转移过程受Na⁺的影响程度与它们的耐盐性相反。（5）钙对三种小麦的总吸钾速率均有促进作用,且随着钙浓度的增加促进作用增加。小麦耐盐能力越强,Ca²⁺对其根系NSCCs转移钾的促进作用也越大。这种促进作用是通过同时提高钾的专性通道吸收和NSCCs转移来达到的;对耐盐能力较弱的小麦来说,Ca²⁺的促进作用则更多的是通过促进钾专性通道转移钾的过程来实现的。（6）钾专性选择性通道和NSCCs的钾转移对细胞膜电位的影响是不一样的:通过专性选择性通道进入根细胞的过程会导致细胞膜的超级化,而通过NSCCs进入细胞的过程则导致细胞膜的去极化。（7）不同耐盐性小麦吸钾过程中在膜电位变化程度方面的差异主要来自于通过NSCCs的钾转移过程,而非通过专性选择性通道的钾转移过程。（8）与耐盐小麦品种相比,盐敏感小麦品种根系的吸钾过程最终导致细胞膜的超级化程度更大。可能原因是在此过程中发生了较多的阳离子通过NSCCs向膜外的转移,即阳离子的外流。（9） Na⁺通过NSCCs进入细胞,倾向于使膜电位向正极方向偏移,外界Na⁺浓度越大,偏移越多,细胞膜去极化程度越大。（10）相对于盐敏感的小麦品种来说,耐盐小麦对低浓度Na⁺不敏感。在高Na⁺浓度下,其维持膜内外电势差的能力较强,这将有利于K⁺的吸收。由此推测,耐盐小麦可能在盐胁迫条件下通过维持较大的细胞膜电势差以保证K⁺吸收,从而实现了体内具有较高的K⁺/Na⁺比。（11）在低盐条件下,Na⁺可以增加耐盐小麦细胞膜的超极化程度,使细胞内外维持较高的电势差,这有利于维持K⁺的吸收;对盐敏感的小麦品种来说,Na⁺使膜电位变化向正极方向偏移,不利于K⁺吸收。高盐条件下,耐盐性强的小麦, NSCCs的钾转移过程所引起的细胞膜去极化程度小,而盐敏感品种的去极化程度大。由此推测,在盐胁迫条件下,耐盐小麦可能在NSCCs转移阳离子的过程中通过维持细胞膜内外较大的电势差,从而具有更强的调节K⁺/Na⁺平衡的能力。（12）在高盐条件下,加抑制剂后盐敏感小麦的细胞膜去极化程度比未加抑制剂时更大。说明高盐胁迫可能使盐敏感小麦通过外向整流型K⁺专性通道发生K⁺的外流,从而使细胞内正电荷减少,形成了较大的膜电势差。（13）钙的加入可以减小由Na⁺所引起的细胞膜的去极化程度,加快膜电位的恢复速度,从而维持较稳定的膜电位,这可能是钙能在一定程度上减轻植物盐害的原因之一。对耐盐小麦来说,Ca²⁺既可抑制Na⁺通过NSCCs向细胞内的转移,又能促进K⁺通过专性通道和NSCCs向细胞内的转移。而对盐敏感小麦来说,Ca²⁺通过减小NSCCs转移Na⁺过程中所造成的细胞膜的去极化,使细胞膜内外维持较大的电势差,从而促进K⁺通道介导的K⁺转移,但对NSCCs的钾转移速率没有影响。更多还原

【Abstract】 Potassium（K⁺） is an essential nutrient and play an important role in many physiological functions of plants. But its relative element sodium is one of the main ions that cause plant damage under saline conditions. To keep a higher intracellular K⁺/Na⁺ ratio is very important for plants to survive in salt stress. Means of a relationship between K⁺, Na⁺ uptake and salt-tolerant. The studies about K⁺ uptake were focused on K⁺ vetors and K⁺ channels before. However, more and more evidences showed that nonselective cation channels （NSCCs） play an important role in many cations absorption. NSCCs mediate current of both sides, participate in K⁺ inflow and outflow. Indicate that NSCCs can regulate intracellular K⁺ content from both aspects. At the same time, NSCCs is also the main pathway for Na⁺ to get into cells. Just because the characters of NSCCs which are absent of K⁺ channels, it can be presumed that NSCCs can not only affect K⁺ uptake directely, but also accommodate intracellular K⁺/Na⁺ ratio by K⁺ and Na⁺ transport of both directions. Whereas the researches about NSCCs’role in K⁺ uptake and influential factors are deficient.The researche route of this paper is to select salt-tolerant and salt-sensitive wheat varieties firstly, and compare their K⁺ uptake conditions. The contribution rate of NSCCs-mediated K uptake of varieties and its influential factors were studied by using of specific inhibitors. Based on this, it is expected that the nutrition uptake and salt-tolerant machanism of different salt-resistant wheat varieties could be revealed from NSCCs aspect, and to provide a reference to cultivate salt-tolerant varieties. The materials were wheat, they are salt-tolerant Shijiazhuang 8, moderate salt-tolerant Yangmai 16 and salt-sensitive Suxu 2. The seedlings were grown hydroponically. K⁺ uptake speed was measured by general kinetics technique. And the plasma membrane potential （MP） was measured by glass micro-electrode. The inhibitors of K⁺ vectors and K⁺ channels were used to separate NSCCs-mediated K⁺ uptake from total K⁺ uptake of plants. The main results were as follows:（1） Salt-tolerance of wheat was concerned to its K⁺ accumulation. Better K⁺ collection led to more resistance to salt stress. At low exoteric K⁺ concentration （showed as [K⁺]ext below）, NSCCs induced K⁺ outflow. K⁺ influx through NSCCs occurred at higher [K⁺]ext.（2） Environment factors can affect NSCCs-mediated K⁺ uptake of wheat roots. Contribution rate of NSCCs-mediated K⁺ uptake decreased at 40℃, indicated that higher temperature was apt to affect NSCCs more than K⁺ channels. At 20℃, the situation was converse. Lower temperature increased the contribution rate and showed preference to affect K⁺ channel.（3） Both cadmium and copper exhibited a concentration-dependent effect on NSCCs-mediated K⁺ uptake of Shijiazhuang 8. Compared with K⁺ channels, NSCCs was less sensitive to Cd²⁺ and more to Cu²⁺. Suggested that these two channel proteins were inhibited by different heavy metal ions through different mechanism.（4） The K⁺ absorption was not suppressed, even promoted by lower [Na⁺]ext. but higher [Na⁺]ext depressed the K⁺ absorption of three wheats significantly.NSCCs-mediated K⁺ uptake of three wheat varieties was affected by Na⁺ differently. NSCCs-mediated K⁺ uptake of Shijiazhuang 8, the most salt-tolerant type, was less influenced, and the contribution rate of its NSCCs up-regulated by higher salt concentration. The relevant of Suxu 2, the most salt-sensitive type, was exquisitely impressed, and the contribution rate of its NSCCs was down-regulated. NSCCs of Yangmai 16, the salt sensitivity of which lies between Shijiazhuang 8 and Suxu 2, were affected moderately. These results indicated that for the varieties with higher salt-tolerance the suppress of Na⁺ to NSCCs-mediated K⁺ influx are usually smaller.（5） Calcium could promote K⁺ uptake of all three wheats. Higher concentration Ca²⁺ had more benefit to salt-resistance. Compared with salt-sensitive wheat, the promotion of Ca²⁺ to NSCCs-mediated K⁺ uptake of salt-tolerant wheat was more significant. For salt-tolerant wheat, the enhancement of Ca²⁺ to K⁺ influx seemed to through both K⁺ channels and NSCCs. But for salt-sensitive wheat, it preferred to affect K⁺ channels.（6） Effects of K⁺ uptake on MP through K⁺ channels and NSCCs were different. K⁺ influx through K⁺ channels led to hyperpolarization, and through NSCCs led to depolarization.（7） For different salt-tolerant wheats, the diverse chang of membrane-potential was due to K⁺ transportation through NSCCs, rather than through K⁺ channels.（8） compared with salt-tolerant wheat, salt-sensitive wheat was more hyperpolarizable upon K⁺ translocation, it might because more cation outflow through NSCCs. （9） Na⁺ uptake through NSCCs led to a positive shift in the changing potential. Higher [Na⁺]ext made more remarkable shift.（10） Compared with salt-sensitive wheat, salt-tolerant wheat was insensitive to lower [Na⁺]ext , allowed little Na⁺ influx, and was good at maintaining potential gradient across plasma membrane at higher [Na⁺]ext, which was helpful to K⁺ uptake. These results meant that salt-tolerant wheat can keep more negative MP to keep K⁺ uptake and help maintaining K⁺/Na⁺ ratio of cells.（11） At low [Na⁺]ext, Na⁺ can increase the hyperpolarization level of salt-tolerant wheat, so that to keep high potential gradient in favor of K⁺ uptake. That might be the reason of impregnabe K⁺ uptake through NSCCs. But for salt-sensitive wheat, Na⁺ induced a positive shift in changing potential, which was harmful to K⁺ uptake, and then reduced K⁺ uptake through NSCCs. At high [Na⁺]ext, the depolarization level of salt-tolerant wheat caused by NSCCs-mediated K⁺ influx was lower than that of salt-sensitive wheat. It can be concluded that salt-tolerant wheat can keep higher potential gradient through NSCCs under salt stress conditions, so as to maintaining higher K⁺/Na⁺ ratio.（12） MP depolarization level of treatment with inhibitors was more significant than without inhibitors at high [Na⁺]ext condition. Suggested that there might be K⁺ out flow through K⁺ outward-rectifying channels of salt-sensitive wheat, with the purpose of reducing positive charge inside cells and keeping potential gradient.（13） Ca²⁺ could help to keep stable membrane potential by making up depolarization caused by Na⁺ and accelerating potential resumption. It seemed to be the reason of lessening of salt toxicity by Ca²⁺. For salt-tolerant wheat, Ca²⁺ could suppress NSCCs-mediated Na⁺ influx and promote K⁺ uptake through both K⁺ channels and NSCC. But for salt-sensitive wheat, Ca²⁺ preferred to promote K⁺ uptake through K⁺ channels than through NSCCs.更多还原