Partial purification and characterization of stomatal phosphoenolpyruvate carboxylase from Vicia faba

Stomatal phosphoenolpyruvate carboxylase (PEPCase EC 4.1.1.31), extracted from abaxial epidermal peels of Vicia faba L. cv. Frühe Weiβkeimige, was partially purified by ammoniumsulfate precipitation, and molecular sieve (Sepharosc S-400) and ion exchange (DEAE-Sepharose) chromatography. The partially purified enzyme, essentially free of a PEPCase isoform existing in mesophyll and epidermal cells, had a specific activity of 300 nkat mg-¹ protein at 25°C. Western immunoblot analysis revealed that the stomatal enzyme had two bands (M_: of 110000 and 112000), crossreacting with PEPCase antibodies raised against PEPCase from Ka-lanchoe daigremontiana . The native molecular mass of the enzyme (467000) points to a tetrameric subunit structure. The temperature optimum was found to be 35°C; cold treatments of PEPCase before assaying were accompanied by inactivation. The energy of activation was calculated to 51 kJ mol-¹. The kinetic behaviour of the enzyme at fixed MgCl₂ concentrations is characterized by a pH optimum between pH 8.0–8.2 with or without 1 m M malate or 5 m M glucose-6-phosphate (Glc-6-P), but a combination of both effectors resulted in a shift of the optimum to pH 7.6. The enzyme showed a pH sensitive inhibition by 1 m M malate and an activation by Glc-6-P. At low pH (6–7), Glc-6-P was able to compensate for the malate induced inhibition of the enzyme. Malate and Glc-6-P both affected K_m(PEP), drastically and influenced V_max at pH 7, but not at pH 8.3. The inhibition constant of malate was determined to be 1.2 m M at pH 7. From the Dixon plot, a competitive inhibition of malate was assumed under defined assay conditions.     

Cell biological mechanism for triggering of ABA accumulation under water stress in Vicia faba leaves

Water stress-induced ABA accumulation is a cellular signaling process from water stress perception to activation of genes encoding key enzymes of ABA biosynthesis, of which the water stress-signal perception by cells or triggering mechanism of the ABA accumulation is the center in the whole process of ABA related-stress signaling in plants. The cell biological mechanism for triggering of ABA accumulation under water stress was studied in leaves of Vicia faba. Mannitol at 890 mmol · kg-1 osmotic concentration induced an increase of more than 5 times in ABA concentration in detached leaf tissues, but the same concentration of mannitol only induced an increase of less than 40 % in ABA concentration in protoplasts. Like in detached leaf tissues, ABA concentration in isolated cells increased more than 10 times under the treatment of mannitol at 890 mmol · kg-1 concentration, suggesting that the interaction between plasmalemma and cell wall was essential to triggering of the water stress-induced ABA accumula     

Effects of turgor potentials of epidermal cells neighbouring guard cells on stomatal opening in detached leaf epidermis and intact leaflets of Vicia faba L. (faba bean)

Leaflets of Vicia faba L. (faba bean) were used to determine whether the mechanical forces resulting from the turgor potentials (Φ_p) of the larger epidermal cells neighbouring guard cells play a significant role in regulating stomatal aperture. When Φ_p, of epidermis and Φ_p of bulk leaflet tissue were compared at midday, Φ_p of epidermis were only 15–25% those of bulk leaflet tissue at all but the most negative leaflet water potentials (Φ). When plants were bagged to increase Φ by reducing vapour pressure differences between leaflets and air, Φ_p of bulk leaflet tissue increased to predawn values, but Φ_p, of epidermis increased to only = 20% of predawn values and stomata opened to their widest apertures. Stomatal apertures were positively correlated with Φ_p of bulk leaflet tissue but they were not correlated with Φ_p of epidermis. Reductions in epidermal Φ_p, began predawn, before stomata were open, and reached minimum values at midday, when stomata were open. We conclude that, in Vicia faba, (1) reduction of Φ_p of epidermal cells begins predawn, reducing the counterforce to stomatal opening that would exist if full epidermal turgor were maintained throughout the day, and (2) changes in Φ_p, of leaf epidermal cells do not play a significant role in regulating stomatal aperture.     

The influence of exogenous proline on the stomatal resistance in Vicia faba

Both the upper and lower stomata of Vicia faba responded to different concentrations of proline supplied either to the 'detached' leaves or sprayed to 'intact' leaves. The stomatal resistance of abaxial surfaces was increased more than that of the adaxial surfaces. A 1-h treatment with 5 m M proline had greater influence than a prolonged treatment. The 'young' leaves responded more than the 'mature' ones to the exogenous proline. A relationship could be demonstrated between endogenous proline, which increased markedly due to exogenous supply, and stomatal resistance.     

蚕豆叶片下表皮ABA结合蛋白提取及分离条件的选择

The abaxial epiderm of Vicia faba L. was chosen to prepare the crude extract of ABA binding protein (ABA-BP). The activity of ABA-BP was dependent on different extraction methods. The specific binding capacity of ABA-BP extracted with 0.5% Triton X-100 (binding activity (B) = 0. 487 nmoL/g protein) was higher than that with extracted cold acetone (0. 325 nmol/g protein) or (NH4)2SO4(0. 223 nmol/ g protein). The activity duration of ABA-BP extracted with Triton X-100 (60% Bmax after 40 h) was longer than that with cold acetone (30% Bmax after 10 h). The ABA-BP activity of binding ABA was sensitive to pH change with an optimum pH of 6.5. The ABA-BP specific binding activity decreased quickly under high concentration of NaC1 ( > 300 mmoL/L), but increased 12% with 5 mmol/L KC1. Some divalent cations like Ca2+ and Mg2 + were required for enhancing the ABA-BP activity. These optimum conditions are primordial for ABA-BP purification.     

洗涤剂对蚕豆根和叶片的作用

研究了家用洗衣粉对蚕豆根生长、细胞分裂和叶片叶绿素含量的影响。结果表明:不同浓度的洗衣粉均能抑制蚕豆根的生长,降低根尖细胞有丝分裂指数,诱发细胞产生高频率的微核且是明显的时间与剂量效应关系和叶绿素含量明显下降。     

Guard cell zeaxanthin tracks photosynthetically active radiation and stomatal apertures in Vicia faba leaves*

Zeaxanthin, antheraxanthin and violaxanthin concentrations in guard cells from sonicated abaxial epidermal peels of Vicia faba were measured from dawn to dusk, and compared with concentrations in mesophyll tissue of the same leaves. Measured changes in guard cell zeaxanthin and violaxanthin concentrations indicate that guard cells operate the xanthophyll cycle throughout the day. Mesophyll tissue had no detectable zeaxanthin at dawn, whereas guard cells had 30–50 mmol mol^?1 chlorophyll a+b. On a chlorophyll basis, maximal zeaxanthin levels were 3–4 fold higher in guard cells than in mesophyll cells. Zeaxanthin concentrations tracked levels of photosynthetically active radiation (PAR) in both mesophyll and guard cells. In the mesophyll, most of the zeaxanthin changes occurred in mid-morning and mid-afternoon. In guard cells, zeaxanthin concentrations changed nearly linearly with PAR in the early morning and late afternoon, and closely tracked PAR levels throughout the day. Guard cell zeaxanthin concentrations were also closely correlated with stomatal apertures. The close relationship between zeaxanthin concentrations and PAR levels in guard cells indicates that zeaxanthin is well suited to function as a molecular photosensor in stomatal movements.     

Light-induced pH and K+ changes in the apoplast of intact leaves

The K⁺-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K⁺ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H⁺ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K⁺ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K⁺, possibly caused by higher apoplastic K⁺, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K⁺ demonstrated that a light-induced decline in apoplastic K⁺ concentration indicative of net K⁺ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H⁺ extrusion into the leaf apoplast due to H⁺-ATPase activity is sufficient for passive K⁺ influx into the leaf cells. Received: 7 March 2000 / Accepted: 12 May 2000     

The kinetics of stomatal responses to VPD in Vicia faba: electrophysiological and water relations models

Abstract. An Ohm's law analogy is frequently employed to calculate parameters of leaf gas exchange. For example, resistance to water vapour loss is calculated as the quotient of vapour pressure difference (VPD) and vapour loss by transpiration. In the present research, this electrical analogy was extended. Steady-state transpiration as a function of VPD, assayed in leaflets of Vicia faba using gas exchange techniques, was compared with steady-state K⁺ current magnitude as a function of voltage in isolated guard cell protoplasts of Vicia faba, assayed using the patch clamping technique in the whole cell configuration. An electrophysiological model originally developed to explain the kinetics of current changes following step changes in voltage across a cell membrane was used to fit the kinetics of transpiration changes following step changes in VPD applied to leaflets of Vicia faba. Following step increases in VPD, transpiration exhibited an initial increase, reflecting the increased driving force for water loss and, for large step increases in VPD, a transient decrease in stomatal resistance. Transpiration subsequently declined, reflecting stomatal closure. By analogy to electrophysiological responses, it is hypothesized that the humidity parameter that is sensed by guard cells is VPD. Two models based on epidermal water relations were also applied to transpiration kinetics. In the first model, the transient increase in transpiration following a step increase in VPD was attributed partially to an increase in the Physical driving force (VPD) and partially to a transient decrease in stomatal resistance resulting from reduced epidermal backpressure. In the second model, the transient decrease in stomatal resistance was attributed to a direct response of the guard cells to VPD. Both models based on water relations gave good fits of the data, emphasizing the need for further study regarding the metabolic nature of the guard cell response to humidity.     

Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba

H₂O₂ is an essential signal in absicic acid (ABA)-induced stomatalclosure. It can be synthesized by several enzymes in plants.In this study, the roles of copper amine oxidase (CuAO) in H₂O₂production and stomatal closure were investigated. ExogenousABA stimulated apoplast CuAO activity, increased H₂O₂ productionand [Ca²⁺]_cyt levels in Vicia faba guard cells, and inducedstomatal closure. These processes were impaired by CuAO inhibitor(s).In the metabolized products of CuAO, only H₂O₂ could inducestomatal closure. By the analysis of enzyme kinetics and polyaminecontents in leaves, putrescine was regarded as a substrate ofCuAO. Putrescine showed similar effects with ABA on the regulationof H₂O₂ production, [Ca²⁺]_cyt levels, as well as stomatal closure.The results suggest that CuAO in V. faba guard cells is an essentialenzymatic source for H₂O₂ production in ABA-induced stomatalclosure via the degradation of putrescine. Calcium messengeris an important intermediate in this process. Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007     

NADPH氧化酶参与水杨酸诱导的蚕豆气孔关闭过程

水杨酸(SA)可以浓度依赖的方式诱导蚕豆叶片的气孔关闭,1～1000μmol·L~(-1)SA所诱导的气孔关闭可以再开放,而10~(-2)mol·L~(-1)的SA导致的气孔关闭则否。质膜NADPH氧化酶抑制剂二亚苯基碘(DPI)可削弱SA作用的45%～60%。表明SA诱导的气孔关闭可能与H_2O_2的产生有关。以H_2O_2荧光探针H_2DCFDA结合显微注射技术直接检测保卫细胞内产生H_2O_2的结果显示,100μmol·L~(-1)SA可引起保卫细胞内荧光素(DCF)荧光快速增强。在DPI存在的情况下,经SA处理的保卫细胞,仅在其叶绿体部位产生H_2O_2,而质膜附近的DCF荧光增强则受到抑制。表明叶绿体可能是保卫细胞内产生H_2O_2的主要部位,质膜NADPH氧化酶也可能参与SA诱导H_2O_2的产生。     

Accumulation of an apoplastic solute in the guard-cell wall is sufficient to exert a significant effect on transpiration in Vicia faba leaflets

Accumulation of recently photosynthesized sucrose in the guard‐cell wall is the empirical foundation for a hypothesis that links the rates of photosynthesis, translocation, and transpiration (Plant Physiology 114, 109–118). Critical assumptions of this hypothesis were tested by use of Vicia faba, an apoplastic phloem loader. Following measurements of the leaflet‐apoplastic‐water volume (by P–V isotherm analysis) and the guard‐cell wall volume (by 3‐D analysis), intact leaflets were fed dilute solutions of mannitol, an impermeant non‐toxic osmolyte. Even at bulk‐leaflet mannitol concentrations that would have only a negligible osmotic effect on stomata, transpiration at constant temperature, water‐vapour pressure, air movement and irradiance was diminished up to 25%, compared with controls. This effect on transpiration, a manifestation of smaller stomatal aperture size, was explained by accumulation of mannitol, up to 350 mol m ^{? 3}, in the estimated aqueous volume of the guard‐cell wall. The conclusion is that mannitol, a xenobiotic with structural similarity to sucrose, can move throughout the apoplast of a transpiring leaflet and accumulate in an osmotically significant concentration in the guard‐cell wall. These data therefore provide support for a new role for sucrose as a signal metabolite that integrates essential functions of the whole leaf. In addition, the results raise questions about the physiological or experimental accumulation of other guard‐cell‐targeted apoplastic solutes such as plant growth regulators, particularly abscisic acid, and ions.     

ABA诱导蚕豆气孔保卫细胞H2O2的产生

以蚕豆叶片下表皮为材料,将荧光探针ＨＰＴＳ导入蚕豆气孔保卫细胞内,利用荧光光谱和激光共聚焦显微技术,检测了ＡＢＡ诱导蚕豆气孔关闭过程中Ｈ２Ｏ２的产生。结果表明,ＡＢＡ和１００μｍｏｌ／Ｌ的Ｈ２Ｏ２可以迅速猝灭ＨＰＴＳ的荧光,ＣＡＴ几乎可完全阻止９μｌ０．２ｍｍｏｌ／Ｌ的ＡＢＡ引起荧光猝灭。因此,ＡＢＡ可以诱导气孔保卫细胞产生Ｈ２Ｏ２,其产生的强度和速度与ＡＢＡ的浓度直接有关,并且推测：这种Ｈ２Ｏ２产生可能是ＡＢＡ诱导气孔关闭过程中信号转导链的一个中间成分。     

Studies on the Micronuclei Induced by Irradiation in Vicia faba

In this experiment ,the remits of observing under the light microscope and electron microscope, and statistical analysis showed that micronucleus had a certain shape and structure. There was a linear correlation between the micronudeated cell frequency and dosage or the chromosomal aberration frequency in the root tip cells. The micronudeated cell frequency changed according to the different periods of time after irradiation. At the same time, micronuclei that might be synchronous, or not, with the main nucleus for the cell cycle were seen. That means it is possible for some micronuclei induced by irradiation to carry on division. Therefore, the advantages of micronucleus test lies not only in simply and effectively evaluating radiation damage to cells but also in exploring the mechanism of radiation genetics in plant.     

Guard-cell apoplastic sucrose concentration--a link between leaf photosynthesis and stomatal aperture size in the apoplastic phloem loader Vicia faba L

In broad bean (Vicia faba L.), an apoplastic phloem loader, the sucrose concentration increases up to approximately 2 mM in the leaf apoplast and up to approximately 150 mM in the guard-cell apoplast during the photoperiod. This high concentration in the guard-cell apoplast results from transpiration and is sufficient osmotically to reduce stomatal aperture size by up to 3 microm or approximately 25% of the maximum aperture size. In this paper, we investigated a parallel and required role for high bulk-leaf apoplastic sucrose concentration, which correlates with high photosynthesis rate. An empirically determined combination of lowered light intensity and lowered CO(2) concentration reduced the photosynthesis rate to nominally one-fifth of the control value without a significant change in transpiration. This reduction in photosynthesis caused the sucrose concentration in the leaf apoplast--the immediate source pool for guard cells--to decrease by 70% (to 0.4 mM). In turn, sucrose concentration in the guard-cell apoplast decreased by approximately 80% (to approximately 40 mM). These results complete the required evidence for a non-exclusive, transpiration-linked, photosynthesis-dependent passive mechanism for the modulation of stomatal aperture size. In an ancillary investigation, hexoses in the bulk-leaf apoplast decreased when photosynthesis was lowered, but their concentrations in the guard-cell apoplast of control plants indicated that their osmotic contribution was negligible.     

Effects of low-level ozone exposure under ambient conditions on photosynthesis and stomatal control of Vicia faba L.

Abstract. Gas exchange rates of 4-week-old faba bean plants were measured after exposure to ozonc (120 μg m³) in an open top chamber for 8 h per day over a period of 2 weeks. The exchange rates were compared with those of control plants. Plants exposed between mid-May and the end of July 1987 showed a minor negative effect on stomatal conductance, while there was no effect on photosynthesis and respiration. Plants exposed between the end of August and early October showed a negative effect on both stomatal conductance and photosynthesis. In addition, the dark respiration rate was slightly increased. It is concluded that ozone can have a direct effect on the stomata as well as on the photosynthetic system and that the stomata are more sensitive than the photosynthetic system.     

Development and accumulation of ABA in fluridone-treated and drought-stressed Vicia faba plants under different light conditions

The effects of fluridone on guard cell morphology, chloroplast ultrastructure and accumulation of drought stress-induced abscisic acid (ABA) were studied in Vicia faba L. plants grown under different light conditions. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. The appearance of defective and undeveloped stomata, and chloroplasts with a destroyed thylakoid membrane system was found in fluridone-treated plants grown at a photosynthetic photon flux (PPF) of 600 μmol m^-2 s^-1. Plants grown at a PPF of 40 μmol m^-2 s^-1 had diminished levels of ABA after imposition of dehydration. Fluridone treatment reduced the level of ABA in both unstressed and dehydrated leaves. Accumulation of ABA in the control plants was considerably reduced when they were exposed to dark periods of 24, 48 and 72 h just before imposition of the stress. Twenty-four hours after the dark treatment dehydration of the leaves resulted in a 3-fold decrease in the level of stress-induced ABA, and 72 h after dark treatment the amount of stress-induced ABA approximated the prestressed values. Fluridone-treated plants failed to accumulate ABA under water stress. In addition to functionally active chloroplasts, well-developed and functional stomata are required for drought stress to elicit a rise in ABA.     

The relations of stomatal closure and reopening to xylem ABA concentration and leaf water potential during soil drying and rewatering

Two tropical tree species, Acacia confusa and Leucaena leucocephala, were used to study the relationships among stomatal conductance, xylem ABA concentration and leaf water potential during a soil drying and rewatering cycle. Stomatal conductance of both A. confusa and L. leucocephala steadily decreased with the decreases in soil water content and pre-dawn leaf water potential. Upon rewatering, soil water content and pre-dawn leaf water potential rapidly returned to the control levels, whereas the reopening of stomata showed an obvious lag time. The length of this lag time was highly dependent not only upon the degree of water stress but also on plant species. The more severe the water stress, the longer the lag time. When A. confusa and L. leucocephala plants were exposed to the same degree of water stress (around –2.0 MPa in pre-dawn leaf water potential), the stomata of A. confusa reopened to the control level 6 days after rewatering. However, it took L. leucocephala about 14 days to reopen fully. A very similar response of leaf photosynthesis to soil water deficit was also observed for both species. Soil drying resulted in a significant increase in leaf and xylem ABA concentrations in both species. The more severe the water stress, the higher the leaf and xylem ABA concentrations. Both leaf ABA and xylem ABA returned to the control level following relief from water deficit and preceded the full recovery of stomata, suggesting that the lag phase of stomatal reopening was not controlled by leaf and/or xylem ABA. In contrast to drying the whole root system, drying half of the root system did not change the leaf water relations, but caused a significant increase in xylem ABA concentration, which could fully explain the decrease of stomatal conductance. After rewatering, the stomatal conductance of plants in which half of the roots were dried recovered more rapidly than those of whole-root dried plants, indicating that the leaf water deficit that occurred during the drying period was related to the post-stress stomatal inhibition. These results indicated that the decrease in stomatal conductance caused by water deficit was closely related to the increase in xylem ABA, but xylem ABA could not fully explain the reopening of stomata after relief of water stress, neither did the leaf ABA. Some unknown physiological and/or morphological processes in the guard cells may be related to the recovery process.     

The apoplastic pool of abscisic acid in cotton leaves in relation to stomatal closure

Suboptimal nitrogen nutrition, leaf aging, and prior exposure to water stress all increased stomatal closure in excised cotton (Gossypium hirsutum L.) leaves supplied abscisic acid (ABA) through the transpiration stream. The effects of water stress and N stress were partially reversed by simultaneous application of kinetin (N⁶-furfurylaminopurine) with the ABA, but the effect of leaf aging was not. These enhanced responses to ABA could have resulted either from altered rates of ABA release from symplast to apoplast, or from some post-release effect involving ABA transport to, or detection by, the guard cells. Excised leaves were preloaded with [¹⁴C]ABA and subjected to overpressures in a pressure chamber to isolate apoplastic solutes in the exudate. Small quantities of ¹⁴C were released into the exudate, with the amount increasing greatly with increasing pressure. Over the range of pressures from 1 to 2.5 MPa, ABA in the exudate contained about 70% of the total ¹⁴C, and a compound co-chromatographing with phaseic acid contained over half of the remainder. At a low balancing pressure (1 MPa), release of ¹⁴C into the exudate was increased by N stress, prior water stress, and leaf aging. Kinetin did not affect ¹⁴C release in leaves of any age, N status, or water status. Distribution of ABA between pools can account in part for the effects of water stress, N stress, and leaf age on stomatal behavior, but in the cases of water stress and N stress there are additional kinetinreversible effects, presumably at the guard cells.Abbreviations and symbols ABA abscisic acid - PA phaseic acid - _w water potential     

SO2对蚕豆根尖细胞微核的诱导作用

应用蚕豆根尖微核试验,对SO2的遗传毒性效应进行了研究。结果表明：一定浓度范围内（0.108-14.00mg/m^3）,蚕豆根尖微核细胞数与SO2浓度间呈正相关,太原市大气污染严重的冬季采暖期根尖细胞微核率明显高于非采暖期;SO2浓度0.604mg/m^3处理24h和48h或2.80-28.00mg/m^3熏气处理4h可使蚕豆根尖中具有微核的细胞数明显增加,结果表明,低浓度SO2较长时间接触或高浓度短期接触均可引起蚕豆根尖细胞遗传物质的损伤,应用蚕豆根尖细胞微核实验可对大气SO2污染进行生物监测。SO2（2.80-28.00mg/m^3）熏气实验中,接触时间延长能导致根尖细胞微核率下降,2.80mg/m^3熏气组下降较快,14.00mg/m^3熏气组下降较慢,研究结果提示,在运用蚕豆根尖微核实验监测环境SO2污染时要考虑蚕豆的染毒方式,避免假阴性结果的出现。