【作者】 刘俊；

【导师】 廖柏寒；

【作者基本信息】 湖南农业大学 ， 生态学， 2010， 博士

【摘要】 通过盆栽试验和水培试验研究了以下内容：(i)红壤镉污染下大豆的生长及主要生理指标在生长周期内的动态变化特征；(ii)镉在大豆植株整个生长周期内的吸收与分配动态特征；(iii)镉胁迫对大豆不同发育阶段生长及生理指标的影响及差异性研究；(iv)镉在大豆幼苗叶中的亚细胞分配、定位及其对幼苗生长的影响；(v)镉在大豆幼苗叶中的累积及其与幼苗生理生化指标的相关性研究；(vi)镉胁迫下不同大豆品种花荚期的生理生态响应。主要研究结果如下：1.红壤盆栽试验结果表明,Cd胁迫对大豆整个生活周期的叶绿素含量、POD活性、SOD活性及MDA含量的影响都是极显著的(ρ<0.01)；短时间、低浓度的Cd胁迫对大豆植株的生长发育有促进作用,高浓度、长时间的Cd胁迫对大豆植株构成明显的毒害作用；大豆株高增长抑制的Cd浓度为1.00 mg·kg-1,远低于大豆生物量的增长受抑制的Cd浓度(2.50 mg·kg-1)；当Cd浓度超过一定水平时,大豆植株生物量和株高的抑制程度与外源Cd浓度呈极显著的正相关(ρ<0.01),对土壤Cd污染具有指示作用,且大豆植株高度与其生物量相比,株高对Cd污染具有更好的指示作用：大豆幼苗期叶绿素含量对镉的敏感性高于开花结荚期和成熟期的敏感性；大豆POD、SOD活性的增加,能在一定程度上减轻Cd胁迫引起的膜脂过氧化造成的伤害作用；在Cd达到2.50 mg·kg-1水平时,植物保护性酶系统活性的提高已经不足以弥补因Cd胁迫对大豆植株造成的伤害；大豆幼苗期和花荚期叶片的POD活性对土壤Cd污染程度具有较好的指示作用,而大豆花荚期和成熟期叶片的SOD活性对土壤Cd污染程度具有较好的指示作用；在Cd胁迫下大豆MDA含量增加,表明细胞膜脂过氧化作用加强。2.红壤盆栽试验结果表明,当外源Cd胁迫浓度一定时,大豆根部Cd含量有着花荚期<幼苗期<成熟期的规律：大豆茎部中Cd含量基本上有着幼苗期<花荚期<成熟期的规律；在幼苗期、花荚期大豆叶中Cd含量随着时间的延长而逐渐提高,但在成熟期大豆叶中Cd含量随着时间的延长有下降趋势；在大豆整个生长周期,当胁迫时间一定时,大豆植株的各器官(根、茎、叶、籽粒、豆荚)中Cd含量随Cd添加浓度的增加而极显著增加,且均表现为乘幂函数关系。3.红壤盆栽试验结果表明,Cd胁迫对大豆植株的生理生态效应随着大豆的生长发育呈现各自不同的特点。大豆植株各发育阶段的MDA含量、生物量和株高的变化趋势没有多大差异,均表现为低浓度的刺激效应和高浓度的抑制效应；但保护性酶系统POD、SOD的活性和叶绿素含量的变化趋势差异显著。在幼苗期,大豆植株的叶绿素含量下降、SOD活性受到显著抑制、POD活性迅速激活,相互协调以缓解Cd的毒性；在花荚期,大豆植株的防御系统得到有效激发,保护性酶POD、SOD的活性急剧升高,叶绿素含量呈上升趋势；在成熟期,因为长时间的Cd毒害,尤其是Cd浓度较高的情况下,大豆植株的SOD、POD活性和叶绿素含量急剧下降。显然,在Cd胁迫下,大豆植株的生长发育以及生理生化指标呈现较为明显的“三个阶段”式变化。4.水培试验结果表明,大豆幼苗叶具有较强的Cd富集能力,叶中累积的Cd含量随着溶液Cd浓度的增加而急剧增加,二者具有明显的幂函数关系,回归方程为：y=10.2x0.308(R2=1.000,n=4)；叶片中Cd大部分储存在细胞壁和可溶性成分中,小部分储存在细胞核、叶绿体和线粒体组分中,尤其是在高浓度Cd胁迫下大量Cd(55.00%)被束缚在细胞可溶性成分中。通过电镜切片可以在细胞壁、叶绿体、细胞核、液泡观察到黑色Cd颗粒沉着。这表明,细胞壁是叶细胞抵抗Cd毒性的第一屏障,细胞壁和可溶性成分是叶细胞储存Cd的主要场所。Cd在叶片细胞器的累积导致细胞间隙扩大、亚微结构受损,尤其是叶绿体的结构损坏,这可能是高浓度Cd抑制幼苗生长的内在原因之一5.红壤盆栽试验结果表明,随着外源Cd胁迫浓度的增加,大豆植株叶中Cd含量显著增加,回归方程为：y=8.76x+4.55 (R2=0.987; n=7, R20.01=0.766,R20.05=0.569)；Cd累积对大豆幼苗的的生长具有低浓度的刺激效应和高浓度的抑制效应;随着叶中Cd富集含量的增加,幼苗叶绿素含量平缓降低,其回归方程为：y=-0.008x+3.300 (R2=0.657, n=7); Cd在大豆幼苗叶中的富集使SOD活性降低；POD活性随着大豆幼苗叶中Cd含量的增加而先增加后降低,二者表现为较明显的抛物线函数关系,回归方程为：y=-0.045x2+5.65x+204(R2=0.578,n=7)；随着叶中Cd含量的增加,大豆幼苗MDA含量的变化呈先下降后上升最终缓慢下降趋势,回归方程为：y=-0.000001x3+0.0001x2-0.003x+0.131(R2=0.804,n=7)。大豆幼苗的株高、生物量、叶绿素含量、POD活性、MDA含量均与幼苗叶Cd含量显著或极显著相关,可以作为大豆幼苗叶Cd累积程度的预警指标。6.大豆五月王和日本青花荚期植株的生物量、株高、SOD、POD活性和叶绿素含量等主要生理生长指标对Cd胁迫的响应存在较大差异。对五月王和日本青植株高度刺激效应最显著的Cd胁迫浓度分别为0.50 mg·kg-1 0.25 mg·kg-1。Cd胁迫对五月王和日本青花荚早期叶绿素合成刺激效应最显著的Cd浓度分别为0.50 mg·kg-1、0.25 mg·kg-1。Cd对五月王和日本青植株SOD活性刺激效应最强的胁迫浓度分别为：2.50 mg·kg-1、1.00 mg·kg-1;在花荚早期,当外源Cd浓度≤0.50 mg·kg-1水平时,随着Cd浓度的增加而日本青植株SOD活性缓慢上升,但五月王植株SOD活性基本上没有变化。这表明,日本青花荚期植株对Cd的敏感性高于五月王花荚期植株的敏感性。更多还原

【Abstract】 A pot experiment and solution culture experiment were conducted to study the following:(ⅰ) dynamic characteristics of main growth and physiological-ecological indicators of soybean plants during the whole growth period under Cd stress; (ⅱ) dynamic characteristics of Cd uptake and distribution in soybean plants during the whole growth period under Cd stress; (ⅲ) the effects of Cd stress on physiological and ecological indicators and their differences in soybean plants at different growth stages; (ⅳ) the effects of Cd on growth of soybean seedlings and subcellular distribution and localization of Cd in soybean leaves; (ⅴ) cadmium accumulation in soybean seedling leaves and correlation between cadmium accumulation and physiological-biochemical indicators;(ⅵ) physiological and ecological responses of two soybean cultivars at flowering-poding stage under Cd stress. The main results were summarized as follows:1. The pot experiments with red soil showed that during the whole growth period of soybean plants, the influences of Cd stress on chlorophyll contents, SOD activities, POD activities, and MDA contents in the leaves were very significant (ρ<0.01). The growth of the plants was enhanced under low concentrations and short time of Cd stress, and restrained under high concentrations and long time of Cd stress. Cd concentration to restrain the plant heights was 1.00 mg·kg-1, which was far lower than that (2.50 mg·kg-1) to restrain the biomass of the plants. When Cd concentration reached a certain level, there was a very significant positive correlation between the restraining effects on biomass and height of soybean plants and Cd concentrations (ρ<0.01), which could be used to indicate soil Cd pollution, especially by using the correlation concerning the plant height. The sensitivity of chlorophyll content to Cd stress was higher at seeding stage than that at flowering-poding and mature stages. Increase of POD and SOD activities could reduce, to some extent, the injury effects of soybean plants due to membrane-lipid peroxidation caused by Cd stress. However, when Cd concentration reached 2.50 mg·kg-1, further increasing activities of plant protective enzyme system did not make up enough for the soybean plant injury caused by Cd stress. The POD activities of soybean at seedling stage and flowering-poding stage, or the activities of SOD at flowering-poding stage and mature stage, could indicate well Cd pollution level in soil. MDA contents in soybean plants increased under Cd stress, implying peroxidation of membrane reinforced.2. The pot experiments showed that under certain Cd concentration stress, the Cd contents in the plant roots at different stages were in the following sequence of flowering-poding stage< seeding stage< mature stage; the Cd contents in the shoots were in seeding stage< flowering-poding stage< mature stage. At seeding stage and flowering-poding stage, the Cd contents in the soybean leaves increased gradually with the time, but decreased at mature stage. During the whole growth period, when the time of Cd stress was certain, Cd contents in all the organs of soybean plants increased with the increasing of Cd concentrations added in soils, and showed power function correlation.3. The pot experiments indicated that the characteristics of physiological and ecological effects were different greatly during the growth of soybean plants under Cd stress. The patterns of MDA contents, biomasses, and heights of soybean plants showed almost the same trends, namely, stimulating effects at low Cd concentrations and inhibitory effects at high Cd concentrations. However, change trends of activities of POD and SOD in the protective enzyme systems and chlorophyll contents in soybean leaves were quite different. At the seedling stage, chlorophyll contents and SOD activities were inhibited obviously, POD activities were activated rapidly, and the mutual coordination of these processes relieved Cd toxicity to soybean plants. At the flowering-poding stage, the antioxidant defense system of soybean plants was excited effectively by Cd stress, resulting in activities of protective enzymes POD and SOD increased rapidly with chlorophyll contents increasing. At the mature stage, SOD and POD activities and chlorophyll contents of soybean plants decreased sharply due to a long-term toxicity of Cd, especially under Cd stress with high concentrations. It was obvious that the indicators of the soybean growth and the physiological and biochemical characteristics behaved a significant pattern of three stages under Cd stress.4. The solution culture experiments showed that the capacity of soybean seeding leaves for Cd accumulation was strong. Cd accumulation in the leaves increased greatly with increasing of Cd concentrations added to the culture solutions, and showed power function correlation (y=10.2x0.308, R2= 1.000, n=4). Most Cd associated with the cell walls and soluble fractions, and a minor part of Cd presented in the nuclear and chloroplast fractions, mitochondria fractions, especially exposure to high Cd concentrations,55.00% Cd were bound in the soluble fractions. Deposited Cd black particles were observed in the cell walls, chloroplasts, nuclei, and vacuoles through electrical microscope slice. This fact indicated that the cell walls of soybean leaves were the first barrier protecting organelles from Cd toxicity, and the cell walls and soluble fractions were the main place for Cd storage. Due to Cd accumulated in the organelles, the intercellular space was enlarged and the subcellular structure was damaged, especially for the chloroplasts. It might be an internal reason of high Cd concentrations inhibiting the growth of plant seedlings.5. The pot experiments showed that Cd contents in soybean leaves increased significantly with increasing Cd stress concentrations, and showed linear function correlation (y=8.76x+4.55,R2=0.987; n=7, R20.01=0.766, R20.05=0.569). Cd accumulated in the seedling leaves showed stimulating effects at low Cd concentrations and inhibitory effects at high Cd concentrations. With increasing Cd accumulated in the seedling leaves, the chlorophyll contents of seedlings decreased slightly, and showed linear function correlation (y=-0.008x+3.300, R2=0.657, n=7). Cd accumulated in the leaves decreased SOD activities. POD activities increased at first and then decreased with increase of Cd accumulated in the leaves, and this correlation could be expressed as a parabola function (y=-0.045x2+5.65x+204, R2=0.578, n=7). With increasing Cd accumulated in the soybean seedlings, MDA contents decreased at first, then increased, decreased slowly finally, and the regression equation was as y=-0.000001x3+0.0001x2-0.003x+0.131 (R2=0.804, n=7). There were significant or very significant correlations between Cd contents in the leaves and the height, biomass, chlorophyll content, POD activity, MDA contents in soybean plants, which could be used as pre-warning indexes for Cd accumulation in the seedling leaves.6. The responses of main physiological and growth indexes, such as biomass, height, SOD, POD activities, and chlorophyll contents, at the flowering-poding stage between two varieties of soybean plants, Wu Yue Wang and Ri Bn Qing, were very different. The most significant stimulating effects of Cd concentrations on the heights of Wu Yue Wang and Ri Ben Qing at flowering-poding stage were 0.50 and 0.25 mg·kg-1, respectively. Those on chlorophyll contents of Wu Yue Wang and Ri Bn Qing at early flowering-poding stage were also 0.50 and 0.25 mg·kg-1, respectively. The most significant stimulating effects of Cd concentrations on SOD activity of Wu Yue Wang and Ri Ben Qing at early flowering-poding stage were 2.50 and 1.00 mg·kg-1, respectively. At early flowering-poding stage, below 0.50 mg·kg-1 of Cd, SOD activity of Ri Ben Qing rose slowly with the increase of Cd concentrations added in soil, but SOD activity of Wu Yue Wang basically changed quite slightly. All the facts above showed that Ri Ben Qing was more sensitive to Cd stress at flowering-poding stage.更多还原