几种氧化酶活性与植物对二氧化硫抗性的研究——植物受SO2污染后有无不可见伤害的商榷

分别用含100和200 ppm SO_2(不致引起可见伤害)的两种亚硫酸溶液,连续40天,每天一次定量喷射大猪屎青(Crotalaria assamica Benth.)后,抗坏血酸氧化酶、多酚氧化酶和过氧化物酶均比对照有较高的酶活性。再用250、275和300 ppm的SO_2(可引起可见伤害浓度)浸泡30秒,以比较其出现可见伤害程度,结果发现,其伤害程度均依次比对照的为重,这一结果表明,经低浓度SO_2喷射过的大猪屎青已有了潜在的伤害,因此认为,大猪屎青对SO_2的反应中,不可见伤害或隐藏伤害是存在的,同时还认为,大猪屎青受到低于可见伤害浓度的SO_2污染后,酶活性的增高(或降低)是不可见伤害或隐藏伤害的标志。     

木本植物对大气重金属污染物耐性的研究

本文在污染现场调查研究、试验研究和单一毒物接触试验的大量数据基础上,论述了木本植物对大气重金属污染物一一铅、镉、铜、锌的耐性。查明了大气重金属污染物引起叶片可见伤害症状需要很高的剂量。糖槭以2000ppm硝酸铅溶液浸泡涂抹枝叶,三天后出现轻度可见伤害症状,叶中铅积累量高达802ppm。旱柳以500ppm氯化镉溶液浸泡涂抹枝叶,叶中积累量达66ppm,十天内未出现可见症状。大气重金属污染物——铅、镉、铜、锌对叶片造成的急性伤害症状是相似的,而与土壤中相同污染物引起的急性伤害具有差别。木本植物对大气重金属污染物的耐性很强,在以大气重金属污染为主的复合污染现场,一些幼龄树木的叶片吸铅量达500—800ppm,超过背景浓度的120倍;吸镉量10—18ppm,超过背景浓度的139倍,吸铜量100—239ppm,超过背景浓度的5—12倍,吸锌量500—700ppm,超过背景浓度的9—20倍只出现较轻的受害症状。在相对清洁区,对幼树叶片进行单一毒物接触试验,一些主要绿化树木的叶片吸铅量达200—600ppm,吸镉量20—60ppm并不出现可见症状。木本植物对大气重金属污染物具有较强的耐性,因而为生物防治大气重金属污染开辟了广阔的前景。     

接骨草叶片硫的积累与转移

用无性繁殖的接骨草幼苗,以不同浓度的SO_2熏气3天和以0.25ppm的SO_2进行不同时间的熏气,测定叶片吸收的SO_2,试验结果表明: 1.接骨草叶片的含硫量和硫的增加量均随SO_2的剂量增加而增加,在SO_2剂量为1.50—1.75ppm·日(平均值1.625ppm·日)以下时,叶片硫的增加量呈直线增加。根据1.625ppm·日剂量时叶片硫的增加量为1.10毫克/克·千重,可推算出SO_2在0.5ppm以下行级浓度时接骨草吸收SO_2的速度和维持吸收的时间。 2.叶片含硫量及硫增加量随叶龄的增加而减少,叶龄愈大,硫的增加量愈小。 3.接骨草叶片停止SO_2熏气以后,叶内含硫量随着时间的延长而逐渐减少,叶龄愈大,其减少的速度愈快。     

植物对二氧化硫的反应和抗性研究——Ⅲ.植物接触二氧化硫和应激乙烯的产生

应用动式熏气装置,研究了SO_2对小麦、水稻、辣椒、蕃茄和紫花苜蓿等植物体内乙烯产生的影响。所有供试植物接触二氧化硫时,如其浓度超过伤害阈值,在出现可见伤害症状之前,体内乙烯产生就显著增加;浓度提高,乙烯量也增多;伤害严重时,并伴有乙烷产生。如果二氧化硫浓度在阈值之下,植物可长期忍受而不发生可见伤害症状的情况下,根据供试植物的反应可分为两类:一类如辣椒,蕃茄,乙烯并不增加,甚至稍有降低趋势;另一类如小麦、水稻和紫花苜蓿,只要接触二氧化硫,就有乙烯增生,不论以后是否出现可见伤害症状。这说明应激乙烯的产生不完全是伤害的后果。从试验结果推测植物体内乙烯的产生可区别为三种水平,即基础乙烯——应激乙烯——伤害乙烯,如伤害严重到一定程度,则还有乙烷的产生,乙烯和乙烷的产生有互相消长的关系。     

烟草叶片受HSO_3~-或SO_2作用时丙烯与丙烷的产生

近年来陆续报道了植物(包括小麦、水稻、辣椒、番茄、苜蓿以及一些葫芦科的植物等)接触SO_2或HSO_3~-以及发生伤害后产生乙烯和乙烷(Bressan等1979,Peiser等1979,李振国等,1980, 刘愚等1980,1982)。最近我们观察到烟草和上述这些植物不同,它的叶片接触SO_2或HSO_3~-和发生伤害后大量产生丙烯和丙烷。就我们所知,植物受SO_2或HSO_3~-作用和伤害过程中产生三碳烃类尚未见报道。     

几种氧化酶活性与植物对二氧化硫抗性的研究——植物本底及受污染后酶活性大小与抗性的关系

对27种不同抗性等级植物本底(未经污染处理的正常植物)多酚氧化酶、抗坏血酸氧化酶和过氧化物酶活性测定结果,植物本底多酚氧化酶活性与抗性有呈负相关的趋势,抗坏血酸氧化酶和过氧化物酶活性大小与抗性不具规律性。 对植物受不同浓度SO_2污染后酶活性变化分析结果看出,蚬木(抗性植物)和汗斑草(敏感植物)在浓度达受阈前,多酚氧化酶、抗坏血酸氧化酶和过氧化物酶均有随浓度的升高而酶活性逐渐增大的趋势,仅是不同的酶其活性高峰在不同浓度梯度中出现迟早不同而己。在污染浓度达受伤阈后,随着浓度的继续增大,酶活性逐渐下降。而白蝉(抗性植物)和大猪屎青(敏感植物)有的酶具有规律性,有的酶不具有这种规律性。 用使可见伤害达50％的SO_2污染汗斑草后4小时(一次污染),过氧化物酶活性为0.34(未受污染的为12.69),仅为未受污染的2.68％,降低了97.32％,但24小时后为7.68,为未受污染的60.52％,比受污染后4小时提高了57.84％,72小时后为8.52,为未受污染的67.13％,比受污染后4小时提高了65.45％。多酚氧化酶亦具有这种规律性。说明二氧化硫对这两种酶的抑制作用是可逆的。     

二氧化硫通过调节谷胱甘肽稳态增强谷子幼苗抗旱性

维持谷胱甘肽稳态是植物适应干旱胁迫的重要方式之一。二氧化硫(SO_2)是一种常见的大气污染物。近年来在植物上的研究发现,外施一定浓度的SO_2能够调节植物响应逆境胁迫的生理过程。但目前关于SO_2对干旱条件下植物谷胱甘肽稳态的调节作用及相关机制仍不清楚。本文以谷子(Setaria italica L.)幼苗为材料,研究一定浓度的SO_2气体暴露对干旱胁迫下谷子谷胱甘肽稳态和干旱适应性的影响。研究结果显示:30 mg·m~(-3) SO_2暴露能够缓解干旱导致的叶片萎蔫症状,增加地上部分生物量积累。与干旱组相比,SO_2+干旱组谷子叶片中的过氧化氢(H_2O_2)含量明显降低(P0.05),叶组织电解质外渗率显著下降(P0.05)。进一步研究发现:干旱胁迫下,SO_2一方面能够提高谷子叶片中的亚硫酸盐还原酶(SiR)、O-乙酰丝氨酸裂解酶(OASTL)活性,诱导SiSiR、SiOASTL基因表达上调,从而增加叶组织中的半胱氨酸(Cys)和还原型谷胱甘肽(GSH)含量;另一方面,SO_2还能够提高谷胱甘肽还原酶(GR)活性及SiGR基因的表达水平,从而维持较高的还原型/氧化型谷胱甘肽(GSH/GSSG)比率,增强叶片抗氧化能力。以上结果表明:干旱条件下,一定浓度的SO_2(30 mg·m~(-3))能够通过调节谷胱甘肽稳态来提高谷子幼苗抗旱性。     

3种阴生地被植物对SO_2胁迫的生理响应及净化能力

该研究以3种阴生地被植物麦冬、虎耳草和紫萼玉簪为研究材料,采用人工模拟熏气方法,测定不同浓度(5.71,11.43,17.14,22.86mg·m~(-3))SO_2胁迫下参试植物的外观受害症状,以及膜质过氧化、保护酶活性、渗透调节物质等生理指标,以叶片吸硫量比较3种植物的净化能力,并采用模糊数学隶属函数与主成分分析法对其抗SO_2能力进行综合评价。结果显示:(1)随着SO_2熏气浓度的升高,3种植物的叶片都有不同程度的受害症状,叶片叶绿素含量、汁液pH值和相对含水量下降,丙二醛含量、叶片相对电导率、可溶性糖、游离脯氨酸含量上升,且其SOD和CAT活性显著增强。(2)隶属函数法和主成分分析法综合评定结果显示,3种地被植物对SO_2抗性能力表现为:麦冬紫萼玉簪虎耳草,与叶片受伤害症状和叶液pH值下降的顺序相反,说明这2个指标可作为简单可行的评价SO_2抗性的重要鉴定指标。(3)3种植物均有一定的SO_2净化能力,其强弱顺序为虎耳草麦冬紫萼玉簪。研究表明,3种阴生地被植物都能够在SO_2胁迫下提高其保护酶活性和渗透调节物质含量,增强其抗硫胁迫和SO_2吸收能力,并以麦冬对SO_2抗性最强,虎耳草对SO_2的吸收能力最强;该试验中最低参试SO_2浓度远远高于城市大气中的实际SO_2浓度,在试验环境下3种阴生植物再都未呈现伤害症状,说明吸收硫能力强的虎耳草和麦冬可以在SO_2污染严重的林下区域大面积应用推广。     

植物抗逆性与光呼吸作用之间的关系

用Na_2SO_3溶液浸泡,可以影响菠菜叶片中超氧物歧化酶的活性。较低浓度(0.1、1、10ppm)的该溶液使酶活性增加,而100ppm的该溶液则降低此酶的活性。同时发现叶片中光呼吸关键酶,即乙醇酸氧化酶活性也随着发生正相关的变化。讨论植物受SO_3~(2-)胁迫后,乙醇酸氧化酶的活性变化与植物抗逆性的产生可能存在密切的关系。     

急性臭氧暴露对大鼠肺部细胞遗传毒性的影响*

目的: 探讨不同浓度臭氧急性暴露对大鼠肺部细胞的遗传毒性的影响。方法: 36只wistar大鼠随机分为对照组(过滤空气暴露)、臭氧暴露组(0.12 ppm、0.5 ppm、1.0 ppm、2.0 ppm、4.0 ppm)共6组,每组6只。以不同浓度的臭氧对大鼠进行动态染毒4 h后,取肺组织并分离单细胞,采用酶联免疫吸附法检测8-羟基脱氧鸟苷(8-OHdG),利用彗星实验、微核试验和DNA-蛋白质交联实验进行DNA和染色体损伤分析。结果: 与对照组相比,肺组织中8-OHdG含量从臭氧暴露浓度为0.12 ppm起即显著增加,在0.5 ppm时达到最高值。随着臭氧暴露浓度升高,彗星拖尾率逐渐上升,且存在明显的剂量-效应关系;DNA-蛋白质交联率有先升高后下降的趋势,且在2.0 ppm时达到最大值;而肺部细胞微核率尽管呈现出上升趋势,但与对照组相比无显著性差异。结论: 急性臭氧暴露在较低浓度(0.12 ppm)时即可导致大鼠肺部细胞的DNA损伤;而在较高浓度(4 ppm)时却未见显著的染色体损伤。     

Alteration of Extracellular Enzymes in Pinto Bean Leaves upon Exposure to Air Pollutants, Ozone and Sulfur Dioxide

Diamine oxidase and peroxidase, associated with the wall in pinto bean (Phaseolus vulgaris L. var Pinto) leaves, can be washed out by vacuum infiltration and assayed without grinding the leaf. The diamine oxidase activity is inhibited in vivo by exposure of the plants to ozone (dose of 0.6 microliters per liter x hour), whereas the peroxidase activity associated with the wall space is stimulated. This dose does not cause obvious necrosis or chlorosis of the leaf. These alterations are greater when the dose of ozone exposure is given as a triangular pulse (a slow rise to a peak of 0.24 microliters per liter followed by a slow fall) compared to that given as a constant square wave pulse of 0.15 microliters per liter for the same 4 hour period. Exposure of the plants to sulfur dioxide (at a concentration of 0.4 microliters per liter for 4 hours) does not result in any change in the diamine oxidase or peroxidase activities, yet the total sulfhydryl content of the leaf is increased, demonstrating the entry of sulfur dioxide. These two pollutants, with different chemical reactivities, affect the activities of the extracellular enzymes in different manners. In the case of ozone exposure, the inhibition of extracellular diamine oxidase could profoundly alter the movements of polyamines from cell to cell.     

EVIDENCE FOR THE REPAIR OF OZONE-INDUCED MEMBRANE INJURY

The increase in [¹⁴C]-2-deoxy-D-glucose uptake by leaf discs 24 hr after fumigation was used as a measure of ozone injury to pinto bean leaves (Phaseolus vulgaris). This method showed that the primary leaves were most affected by ozone when plants were 10 to 12 days old, which coincided with the time of maximum leaf necrosis. However, 8-day-old plants, which had no visible leaf injury, still showed a higher uptake rate than controls, indicating that injury occurred at the cellular level. In these younger plants, uptake was shown to return to normal over a 5 day period. Moreover, the rate of this “repair” was retarded by cold or continuous darkness, enhanced by continuous light, and very markedly increased by glucose applied to the leaves. The timing of the glucose application was not critical, nor did H₂O or mannitol have an effect. The results suggest that ozone injury at the cellular level can be repaired by energy-dependent processes so that necrosis of the leaf tissue does not occur. These experiments also show that conditions and treatments after ozone exposure can alter the degree of ozone injury.     

THE INDUCTION OF LEAF TUMORS BY AGROBACTERIUM TUMEFACIENS

Using carborundum as an abrasive and light rubbing with a culture of Agrobacterium tumefaciens, leaves of various species of bean and tobacco develop tumors on the leaf lamina. The induction of these tumors requires wounding, the presence of a virulent strain of the bacterium and is due to the bacterium, not substances released into the bacterial culture medium during growth. Observations of the histology and cytology of these tumors on the primary leaves of pinto bean show no significant differences from the more commonly studied stem tumors. The tumors on pinto beans first appear as chlorotic nests of dividing cells which gradually accumulate chlorophyll, eventually becoming dark green in color as opposed to the surrounding leaf tissue which is completely chlorotic at this stage. Tumor development is enhanced by a dark period following inoculation while growth of the leaf is essentially stopped. The tumors thus exhibit a pattern of growth and development independent of that of the normal leaf. The number of tumors obtained on pinto bean leaves was found to depend on the concentration of bacteria in the inoculum and on the age of the plants. A sharp peak in response was observed at about 7 days from planting. Best results were obtained by adding the bacterium at the time of wounding. The tumors were shown to differ from IAA-induced leaf proliferations with respect to their point of origin on the leaf, morphology, physiology and development.     

Bioassay and attributes of a growth factor associated with crown gall tumors

An improved bioassay is described for a factor that promotes tumor growth which was first obtained from extracts of pinto bean leaves with crown gall tumors. Sixteen primary pinto bean leaves per sample are inoculated with sufficient Agrobacterium tumefaciens to initiate about 5 to 10 tumors per leaf and treated with tumor growth factor at day 3 after inoculation. The diameters of 30 to 48 round tumors (no more than 3 randomly selected per leaf) are measured per test sample at day 6. Mean tumor diameter increased linearly with the logarithm of the concentration of tumor growth factor applied. The tumor growth factor was separated by column chromatography from an ultraviolet light-absorbing compound previously reported to be associated with fractions having maximal tumor growth factor activity. Partly purified tumor growth factor showed no activity in a cytokinin bioassay or an auxin bioassay, and negligible activity in gibberellin bioassays. Representatives of these three classes of growth factors did not promote tumor growth. Extracts from crown gall tumors on primary pinto bean leaves, primary castor bean leaves, Bryophyllum leaves, carrot root slices, and tobacco stems showed tumor growth factor activity, whereas extracts from healthy control tissues did not. Extracts from actively growing parts of healthy pinto beans, Bryophyllum, and tobacco, however, showed tumor growth factor activity. Tumor growth factor is proposed to be a normal plant growth factor associated with rapidly growing tissues. Its synthesis may be activated in nongrowing tissues by infection with Agrobacterium sp.     

OZONE-INDUCED MEMBRANE PERMEABILITY CHANGES

The effect of 0.5 ppm ozone for 0.5-1 hr on plant cell membrane permeability was ascertained. Permeabilities to both water and solutes were estimated by measuring leaf disc weight changes and following tritiated water and ⁸⁶Rb fluxes. Measurements were made immediately after ozone exposure and 24 hr after exposure. The reflection coefficient, σ, an index of solute permeability, decreased in ozone-treated primary leaves of pinto bean (Phaseolus vulgaris). The latter indicates an increase in membrane solute permeability or internal solute leakage. Water and THO flux estimates both indicated a decrease in membrane permeability to water; both the hydraulic conductivity (L_p) and the water diffusional coefficient (L_D) apparently decreased, an anomaly which is discussed. These data indicate that ozone has a direct effect on membrane function by altering permeability characteristics. We assume from these data that cell membranes are primary target sites for ozone injury.     

Effect of ozone on ribosomes in pinto bean leaves

A study was made of cytoplasmic and chloroplast ribosomes in the primary leaves of pinto bean plants exposed to ozone. The isolated ribosomes were analysed by sucrose density gradient. Ozone at the levels of 0·35 ppm for 20–35 min does not change the concentrations of various sedimenting particles of the cytoplasmic ribosomes. Ozone at similar levels, however, specifically decreases the population of chloroplast ribosomes per unit fresh weight of leaves. The distribution pattern of these chloroplast ribosomes is characterized by the low concentration of the fast-sedimenting polysome particles concomitant with the low magnitude of other slow-sedimenting components. The kinetics of ribosome populations during leaf growth demonstrates that ozone does not influence the daily levels of different ribosomal components of cytoplasmic ribosomes. However, ozone prematurely decreases the concentrations of polysomes and other components of chloroplast ribosomes below control level at the early stage of leaf development. These findings are discussed to explain initiation of the premature senescence caused by ozone.     

Ovarian cyclicity and serum progesterone and luteinizing hormone in fine-wool ewes supplemented with alfalfa or pinto beans

Effects of season and supplementation on the incidence and rate of ovulation and hormone profiles in multiparous, nonlactating ewes were investigated under range and drylot conditions during anestrus (February through August). Ninety ewes received one of six nutritional treatments: 1) range forage, 2) range forage plus 0.33 kg.hd(-1).d(-1) pinto beans, 3) 0.45 kg.hd(-1).d(-1) alfalfa pellets, 4) dry lot plus 1.33 kg.hd(-1).d(-1) prairie hay only, 5) pinto beans or 6) alfalfa pellets at rates used on the range and prairie hay. Supplemented ewes were heavier (P < 0.05) than ewes fed range forage or prairie hay during most of the study. Seasonality of ovulation, in terms of incidence and rate, was not affected (P > 0.40) by supplementation in range ewes. The ovulation rate tended to be highest in prairie hay plus pinto bean drylot ewes in February (P = 0.21) and in prairie hay plus alfalfa pellet and prairie hay plus pinto bean ewes in March (P = 0.13) compared with prairie hay ewes. Generally, seasonality of ovulation was not influenced by supplementation (P > 0.40). The incidence of ovulation approached zero for drylot ewes in April, May, June and July, while it was 43, 27, 35 and 21% for range ewes, in those same months. Supplementation did not affect serum progesterone during the estrous cycle. Luteinizing hormone (LH) concentrations were similar (P > 0.50) among drylot treatment groups before a 50-ug gonadotropin releasing hormone (GnRH) challenge. Pinto bean supplementation enhanced serum LH response to GnRH (P < 0.10).     

Effects of SO(2) on Stomatal Metabolism in Pisum sativum L

Pea (Pisum sativum L. cv `Little Marvel') plants were exposed to SO₂ for short term (3 hours) and long term (2 days) at 0.2 and at 0.5 microliter per liter (ppm) levels. The effect of this treatment on the activity of phosphoenolpyruvate carboxylase, NAD- and NADP-malate dehydrogenases, and alanine aminotransferase from epidermis and whole leaves was investigated. Short-term exposure to SO₂ at 0.2 or 0.5 ppm decreased the activity of the carboxylase and the dehydrogenases in the epidermis. In contrast, the activity of the same three enzymes increased in whole leaves with either short- or long-term exposure to SO₂. Alanine aminotransferase in epidermis or whole leaves was not much affected by short-term exposure, but the epidermal activity was decreased and whole leaf activity was increased with long-term exposure. SO₂ exposure which was initiated prior to illumination decreased the free thiol content of both epidermis and of whole leaf. Net photosynthesis was reversibly inhibited by long-term exposure to SO₂ at 0.5 ppm. No effect of 0.5 ppm SO₂ on stomatal conductance was detectable after 3 hours. Stomatal conductance appeared to decrease after longer exposure times (2 days) at 0.5 ppm.     

Chlorophyll fluorescence assay for ozone injury in intact plants

A chlorophyll fluorescence induction (Kautsky effect) assay predicted ozone-induced injury in bean leaves (Phaseolus vulgaris) at least 20 hours before any visible sign of leaf necrosis. The extent of injury, which could be predicted during exposure to ozone, depended on concentration, exposure time, and leaf development stage. Much more injury occurred in light than in darkness and long exposures to lower ozone concentrations were more injurious than brief exposures to higher ones. The first detectable effect was on the photosynthetic water-splitting enzyme systems, followed by inhibition of electron transport between the photosystems. The fluorescence assay provides a simple, rapid, nondestructive method for observing effects of ozone on plants.     

On Acute Injury and Relative Resistance to Sulfur Dioxide of Trees and Shrubs for Greening

In order to select trees and shrubs resistant to SO2 and to provide information concerning acute injury, fumigation experiments were carried out on 71 species common to the region of Shanghai at three seasons in the phytotron. Two identical dynamic fumigation cabinets were used. The branches detached from plants were fumigated for 6 hours at concentrations ranging from 0.5 to 5 ppm. The patterns of acute leaf injury and the seasonal variabilities of resistance to SO2 were observed and are described. Based on the estimated threshold value of leaf injury, the relative resistance to SO2 of the tested trees and shrubs can be grouped into 5 categories. The order of resistance is in the main in accordance with the field experience.