Abstract:

Soil salinization and alkalization causes serious losses in agricultural and husbandy productivity, which is also one of ecological crisis that humans face. It has significance in improving and using the degeneration saline-alkaline grassland, that study the physiological responses characteristic of plant to salt and alkali stresses, and improve the growth ability of plants and seed productive. Plant hormones are one of the main signal transduction components of plant responding to stresses. The hormones contents will change with stress concentrations. Application of exogenous hormones can alleviate the damage of abiotic stress on plant. Furthermore, plant hormones are important signal substances of flower formation. IAA, ABA and GA₃ play key roles in controlling the flower formation and seed production of plants. In this paper, we used annual crop and perennial pasture, wheat (Triticum aestivum) and Leymus chinensis as the experiments materials, and studied the effects of salt and alkali stresses on the seedlings growth; and alleviated function of ABA on both stresses; and application of plant hormones on improvement of sexual and vegetative reproduction of L. chinensis. The main results and conclusion were as following:
1. Wheat had the different physiological responses mechanisms between salt and alkali stresses. The slat (NaCl:Na₂SO₄=9:1,pH=6.27~6.45) and alkali (Na₂CO₃:NaHCO₃=9:1,pH=9.10~9.17) stresses with 0, 40, 80 and 120 mmol·L^-1 concentrations were applied on 1 week wheat seedling.200 mL treatment solution was used every day per pot (the diameter of the pot was 21 cm, and planted 15 seedlings) for 9 days until the leaves turned yellow under alkali stress with the height stress concentration. The results showed that wheat had different physiological response characteristics to slat and alkali stresses. Wheat significantly accumulated inorganic ions (Na⁺ and Cl^-) and organic solutes (soluble sugars and proline), but not accumulated organic acids under salt stress. Under alkali stress, wheat accumulated Na⁺, organic acids, soluble sugars and proline, but not accumulated Cl^- and H₂PO^-₄ as inorganic ions. The accumulation of Cl^- and organic acids was the main difference of wheat in the physiological responses and adaptive mechanisms to salt and alkali stresses, respectively. Organic acids were the special solutes that wheat responded to alkali stress.
We concluded that malate and citrate were the main OAs components of wheat seedling through determining the contents of organic acids components (OAs) malic acid, citric acid, acetic acid, oxalic acid, succinic acid and formic acid in wheat shoots and roots, but the contents and increments of OAs between shoots and roots were different. All OAs components contents in shoots were higher than in roots, but the increment in roots were higher than that in shoots. With increasing alkalinity, malate and citrate increased in shoots, but in roots they increased first then decreased. The results indicated that different organs of wheat seedlings had different response to alkali stress.
2. Exogenous plant hormones-abscisic acid could remit the adverse influence of salt and alkali stresses on wheat seedlings effectively. 100 mmol·L^-1 was used in this experiment. ABA of 50 and 100 μmol·L^-1 were set at 1, 4, 7 and 10 days after stresses. The results showed that foliar application of ABA can alleviate the adverse effect of salt and alkali stresses on wheat seedlings. Because of the different responding mechanisms of wheat seedlings to salt and alkali stresses, the alleviation mechanisms of ABA on salt and alkali stresses were also different. ABA can decrease the accumulation of Na⁺ caused by salt stress, and increase the ratio of K⁺/Na⁺ and Ca²⁺/Na⁺ to improve the salt resistance of wheat. Under alkali stress, application of ABA can increase the synthesis of organic solutes, such as soluble sugars, organic acids components contents in shoots to decrease the adverse effect of alkali stress on wheat. The lower concentration (50 μmol·L^-1)of ABA had better mitigation effects than the higher concentration(100 μmol·L^-1).
3. L. chinensis had different physiological responses mechanisms between salt and alkali stresses, rhizomes played key roles. Fifty, 100, 150, 200 mmol·L^-1 were used in this experiment, because L. chinensis was perennial pasture, which had higher resistance to drought, cold and saline-alkaline. 500 mL treatment solution was used every day per pot (the diameter of the pot was 21 cm, and planted 7 seedlings) for 10 days. Biomass, photosynthesis, inorganic ions and organic solutes were determined. The results showed,under salt stress, the inorgaic ions such as Na⁺ and Cl^- were the main osmotic ajustment substances of L. chinensis, the osmotic regulation of organic solutes such as soluble sugars, prolines and organic acids were little. Na⁺ and Cl^- accumulated in L. chinensis distributed mainly in rhizome, and NO₃^- contents were the lowest in rhizome. The capacity of containing toxic ions in rhizomes protected the normal growth of other organs, and avoided adverse effect of salt stress on them. Under alkali stress, L. chinensis accumulated Na⁺, and soluble sugars, proline, and organic acids to involve in the osmotic ajustment. The rhizome contained higher Na⁺ and Cl^- and lower NO₃^- at lower concentration of stresses. When the alkaline concentration increased, the contents of Na⁺, soluble sugar, proline, and organic acids in stems and leaves were increased sharply, which indicated that this concentration (200 mmol·L^-1) caused serious damage on L.chinensis, the synthesis of organic solutes was the procuct of damage. Rhizome played key roles in L. chinensis,responding to the alkali stress at a certain concentration.
4. Exogenous plant hormones affected the vegetative propagation of L. chinensis, significantly. Randomized block design was used in an artificial L. chinensis grassland. 0.5 m was set as guard row among experimental plots (12 m²). Gibberellin (GA₃,75 mg·L^-1), auxin (IAA,75 mg·L^-1)and kinetin (KT,150 mg·L^-1)were used at dusk and at floret differentiation stage of L. chinensis. Samples with 1 m×1 m and 20 cm depth were got every 30 days to investigate the number of buds and seedlings. The endogenous hormone content was determined by HPLC method. The heading number and heading percentage were investigate the next year. The results showed that GA₃ significantly increased the number of rhizome buds, node buds and rhizome shoots at the end of growth season, decreased endogenesis IAA and ABA contents, then significantly increased the heading number and heading percentage of unit area next year. We concluded that the rhizome buds and rhizome shoots were the main components of heading shoots next year. The decreases of endogenesis IAA and ABA were the foundation of inducing differentiation on reproductive buds. Although exogenous IAA and KT can increase the daughter shoots came from rhizome and rhizome node, neither of them increased the heading numbers and heading percentage of L. chinensis population next year. The reason was that exogenous IAA and KT induced the increase of ABA contents, and decreased IAA/ABA, and had no significant active effect on reproductive buds differentiation. Therefore, exogenous gibberellin had significant effects on vegetative propagation of L. chinensis, comparing with auxin and kinetin.
5. Exogenous plant hormone could promote sexual propagation ability of L. chinensis effectively. GA₃, IAA and KT of 75 mg·L^-1 and 150 mg·L^-1 concentration were sprayed at greenup, tillering and jointing stage separately. The reproductive shoots were signed at full-bloom stage to investigate the heading number and percentage, plant height, ear length, the number of spikelet, flora and seeds, the weight of panicle and seeds, then calculated the setting percentage and seed yield. The results showed that there was no significant effect of GA₃, IAA and KT on heading number and heading percentage when they were sprayed at the three stages. This was related to the process of spikelets differentiation. L. chinensis had completed the spikelets differentiation in this time. On greenup stage, GA₃ treatment increased seed number, seeding percentage, weight of spikes and seeds, but there was no significant effect of GA₃ treatment on tiller stage. On shooting stage, GA₃ could increasing the number of florets and seeds per spike significantly, and then increasing the weight of spike and seeds per spike and seed yield. So we concluded that GA₃ accelerated the florets differentiation of L.chinensis. There was no positive effect of IAA on the traits of sexual reproduction number characters when it sprayed on re-growth stage, tiller stage and shooting stage. The effect of KT that sprayed on re-growth stage on seeding of L. chinensis was similar to that on tiller stage. They didn't change the seeding percentage, but increased the seed numbers and spike weights, which played active function in seed filling and development. On shooting stage, KT could increasing the number of florets and seeds per spike significantly, and then increasing the weight of spike and seeds per spike and seed yield. But there was no difference between different concentrations. The regulation of plant hormone on growth and reproduction was correlated with the treatment time, concentration and action mechanisms of plant hormone.

Key words: Salt and alkali stress, Wheat, Leymus chinensis, Physiological response, Exogenous plant hormones

摘要：

盐碱胁迫给农牧业生产造成了严重的损失，是人类面临的生态危机之一。研究植物对盐碱胁迫的生理响应特点，提高植物生长能力和产量对改良和利用退化盐碱草地具有重要意义。植物激素是植物响应胁迫的信号转导的主要成员，其含量随着胁迫浓度变化而变化，且外用植物激素可缓解非生物胁迫对植物的伤害。此外，植物激素也是成花的重要信号物质，植物激素如IAA 、ABA和GA₃等对植物成花和结实具有一定的作用。针对东北盐碱土壤的现状，本论文以一年生粮食作物小麦（Triticum aestivum）和多年生牧草羊草（Leymus chinensis）为材料，分别研究了盐胁迫和碱胁迫对小麦幼苗和羊草幼苗生长发育的影响、外源ABA在提高小麦耐盐碱性中的作用以及外源植物激素对羊草有性及无性繁殖的影响。主要研究结果和结论如下：
1. 小麦幼苗对盐胁迫和碱胁迫表现为不同的生理响应机制。将浓度为0，40，80，120 mmol·L^-1的盐胁迫（NaCl∶Na₂SO₄=9∶1，pH=6.27~6.45）和碱胁迫（Na₂CO₃∶NaHCO₃=9∶1，pH=9.10~9.17）分别应用在生长1周的小麦幼苗上，每天每盆（21 cm 直径盆栽，沙培法，每盆15棵幼苗）用250 mL处理液分2次透灌，连续处理9 d，直至最高胁迫浓度的幼苗叶片变黄为止。研究表明，小麦在盐胁迫和碱胁迫下表现出不同的生理响应特征。在盐胁迫下，小麦大量积累Na⁺、Cl^-等无机离子及脯氨酸和可溶性糖等有机溶质，而未积累有机酸；在碱胁迫下，小麦积累大量Na⁺和有机酸、可溶性糖和脯氨酸，而未积累Cl^-和H₂PO^-₄等无机阴离子。有机酸和Cl^-的积累是小麦对盐、碱胁迫生理响应机制和适应对策的根本区别所在，其中有机酸是小麦对碱胁迫的特殊响应物质。
在试验1基础上进一步对小麦茎叶和根中有机酸组分和含量进行分析，在小麦幼苗内检测出苹果酸、柠檬酸、乙酸、草酸、琥珀酸和甲酸，其中从有机酸组分含量分析上发现苹果酸和柠檬酸是小麦幼苗抵御碱胁迫伤害的最主要有机酸成分；有机酸组分在茎叶和根中的含量和变化趋势不同，茎叶中的有机酸含量虽然明显高于根，但碱胁迫后根中有机酸增加量却明显大于茎叶；随着碱胁迫的增强，茎叶中的苹果酸和柠檬酸显著增加，在根中则呈现先上升后下降的趋势。这表明小麦幼苗不同器官对碱胁迫的响应也不同。
2. 外源植物激素-脱落酸能有效缓解盐碱胁迫对小麦幼苗的不利影响。根据试验1中120 mmol·L^-1碱胁迫浓度对小麦的生长影响较大，本试验将盐碱胁迫浓度降低为100 mmol·L^-1。同时配置浓度为50 μmol·L^-1和100 μmol·L^-1的脱落酸（ABA）溶液，分4次在盐碱胁迫处理的第1，4，7，10 d，喷到小麦叶片上，以成滴为准。结果表明：叶面喷施ABA可有效缓解盐胁迫和碱胁迫对小麦的伤害。小麦对盐胁迫和碱胁迫的生理响应机制不同，ABA在这些胁迫上的缓解机制也不同，ABA通过降低盐胁迫所引起的钠离子的积累，提高K⁺/Na⁺和Ca²⁺/Na⁺的比值来达到缓解盐胁迫对小麦的伤害作用；ABA主要通过增加响应碱胁迫的有机溶质的合成，如可溶性糖、柠檬酸和琥珀酸等含量，来缓解碱胁迫对小麦的伤害作用，且低浓度（50 μmol·L^-1）的缓解效果好于高浓度（100 μmol·L^-1）。
3. 羊草幼苗对盐胁迫和碱胁迫的生理响应机制不同，根茎在羊草幼苗抵御一定浓度盐碱胁迫过程中发挥重要作用。由于羊草是多年生牧草，具有较强的耐旱、耐寒和耐盐碱性，因此设置的盐碱胁迫浓度较高，为50，100，150和200 mmol·L^-1 4个梯度。每天每盆（21 cm 直径盆栽，沙培法，每盆7棵幼苗），用500 mL处理液分3次透灌，连续处理10 d，测生物量、分蘖能力、光合、无机离子和有机溶质含量。结果表明：在盐胁迫下，羊草积累以Na⁺和Cl^-为主的无机离子，参与渗透及离子平衡调节，脯氨酸、可溶性糖和有机酸等有机溶质作为渗透溶质的调节作用较小；在所有器官中，根茎含有相对高的Na⁺和Cl^-等盐离子含量及最低的硝酸根离子含量，根茎这种对盐离子的容纳及营养离子的贡献作用，避免了盐胁迫对其他器官生长的伤害，保护了其他器官的正常生长。在碱胁迫下，羊草积累Na⁺的同时，更多的积累脯氨酸、可溶性糖和有机酸等有机溶质来参与碱胁迫下的渗透调节，低浓度时根茎含有相对较高的Na⁺含量和较低的硝酸根含量，而当碱胁迫浓度增高时，Na⁺流向茎和叶内的含量增多，进而导致茎叶内合成可溶性糖、脯氨酸和有机酸的含量高于其他器官，茎和叶内盐离子和有机溶质大量增加，表明高浓度（200 mmol·L^-1）的碱胁迫使羊草生长受到严重损害，有机溶质的合成是胁迫伤害的产物。在一定碱胁迫浓度下，根茎在羊草幼苗适应生长中发挥重要作用。
4. 外源植物激素显著影响羊草的无性繁殖能力。选择羊草人工草地，采用随机区组设计划分试验小区（12 m²），小区间设0.5 m保护行。在羊草小花分化期，选择傍晚向叶面喷施赤霉素（GA₃，75 mg·L^-1），生长素（IAA，75 mg·L^-1），激动素（KT，150 mg·L^-1），4次重复。处理后每间隔30 d破坏性取样1 m×1 m，深20 cm，统计各类芽和苗数量。同时采用高效液相色谱方法测试根茎顶端芽的内源激素含量，次年调查抽穗数和抽穗率。结果表明，外源GA₃处理下，羊草生长季末的根茎顶端芽、根茎节间芽和根茎顶苗的数量显著增加，内源IAA（生长素）和ABA的含量显著降低，翌年单位面积羊草的抽穗数和抽穗率显著提高，由此可以推出根茎顶端芽及根茎顶苗是翌年羊草种群生殖枝的主要来源，内源IAA和ABA含量的下降是促进花芽分化的基础。外源IAA和KT（激动素）处理下，羊草根茎苗和节间苗数虽有增加，但翌年羊草种群的抽穗数和抽穗率没有显著变化，是因为外源IAA和KT均导致ABA含量的增加，从而IAA/ABA的比值显著下降，对花芽分化没有显著促进作用。因此，与生长素和激动素相比，外源赤霉素对羊草无性繁殖具有显著影响。
5. 外源植物激素可有效促进羊草有性繁殖能力。与试验4应用相同试验小区设计，分别在羊草返青期、分蘖期、拔节期向叶片喷施75 mg·L^-1和150 mg·L^-1的赤霉素（GA₃）、生长素（IAA）和激动素（KT）。于盛花期选择同天开花的生殖枝挂牌标记，调查抽穗数并计算抽穗率，观测株高、穗长、每穗的小穗数、总小花数、结实数，并计算结实率，记录单穗重和粒重，换算种子产量。结果显示返青期、分蘖期和拔节期分别喷施GA₃、IAA及KT等3种激素对当年羊草的抽穗数与抽穗率均没有显著影响，这与羊草的幼穗分化时期和进程有关，此时期羊草已完成了幼穗分化。返青期施用GA₃处理显著提高了羊草的结实数、结实率、穗重和粒重；分蘖期施用GA₃对有性繁殖数量性状无显著影响；拔节期喷施GA₃通过显著地增加羊草每穗小花数和结实数而显著增加穗重、粒重及种子产量。GA₃对羊草的促进作用受施用时间和植物生长状态影响，GA₃处理后，不仅显著提高了结实率，而且大大降低了结实率的变异幅度，使羊草抽穗整齐，开花集中，授粉充分，结实率较高。返青期、分蘖期和拔节期施用IAA对羊草有性生殖数量性状均无显著促进作用。返青期和分蘖期喷施KT对羊草有性繁殖的影响结果相似，虽然未改变结实率，但是显著提高了羊草每穗结实数和粒重，对羊草的种子灌浆及发育具有明显的促进作用；拔节期喷施KT通过显著增加羊草每穗小花数和结实数而显著增加穗重、粒重及种子产量，不同KT浓度间无显著差异。外源植物激素对羊草有性结实的影响与其施用浓度、施用时间和生理特性有关。

关键词: 盐碱胁迫, 小麦, 羊草, 生理响应, 外源植物激素