Regulation of enzyme levels by phytochrome in mustard cotyledons: Multiple mechanisms?

Phytochrome controls the appearance of many enzymes in the mustard (Sinapis alba L.) cotyledons. The problem has been whether the effect of phytochrome on the appearance of enzymes in this organ is due to a common initial action of P_fr, e.g. due to the liberation of a second messenger. We have compared the modulation by light (phytochrome) of the appearance of phenylalanine ammonia lyase (PAL)⁺ and ribulosebisphosphate carboxylase (Carboxylase)⁺. PAL becomes detectable in the mustard cotyledons at 27 h after sowing while Carboxylase starts to appear only at 42 h after sowing (starting points, 25° C). The starting points cannot be shifted by light. As a major result, in the case of PAL the inductive effect of continuous red light (given from the time of sowing) remains fully reversible by 756 nm-light up to the starting point (27 h after sowing) while with Carboxylase full reversibility in continuous red light is lost at approximately 15 h after sowing. While the induction of Carboxylase is already saturated at a very low level of P_fr (e.g. continuous 756 nm-light saturates the response) and does not depend on irradiance (e.g. continuous 675 mW m^-2 red light and 67.5 mW m^-2 red light lead to the same time course), PAL induction is a graded response over a wide range of P_fr doses and depends strongly on the fluence rate (high irradiance response, HIR). It is concluded that PAL induction and Carboxylase induction are not only separated in time but differ in every regard except that both responses are mediated by phytochrome.The present data support the previous conclusion that the specification of the temporal and spatial pattern of development is independent of phytochrome even though the realization of the pattern of development can only occur in the presence of phytochrome (P_fr). It seems that there is no feedback from pattern realization to pattern specification.Abbreviations P_fr the far-red absorbing, physiologically active form of phytochrome - P_r the red absorbing physiologically inactive form of phytochrome - P_total [P_r]+[P_fr] - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Carboxylase ribulosebisphosphate carboxylase (EC 4.1.1.39)     

Elicitor induction of furanocoumarin biosynthetic pathway in cell cultures ofRuta graveolens

Treatment with an autoclaved culture homogenate of the yeastRhodotorula rubra induces rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins in cell cultures ofRuta graveolens (L). The increased accumulation is preceeded by an induction of enzymes of the biosynthetic pathways. In the case of furanocoumarins induction was shown for phenylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4-CL) and S-adenosyl-l-methionine: xanthotoxol O-methyltransferase (XOMT). For PAL and 4-CL time courses of induced activity showed an early maximum, 8–12 h after treatment, whereas XOMT was found to reach its maximum later, about 36–42 h after treatment. The elicitor dose-response curve showed saturation at an elicitor concentration of 1%. At any time during the whole culturing period cells responded to elicitiation but the maximum enzyme activities induced were lower at the late stages. Experiments with different suspension culture strains, a shoot teratoma culture and hydroponically grown sterile photomixotrophic plants were performed to assess the influence of differentiation on constitutive activities of these enzymes and their inducibility by elicitation. Constitutive furanocoumarin accumulation was positively correlated with the level of differentiation. Although induction of PAL, 4-CL and XOMT activity always accompanied induced furanocoumarin accumulation no absolute correlation existed between induced enzyme activities and the induced product level or relative product increase.Abbreviations 4-CL 4-coumarate:CoA ligase - COMT S-adenosyl-l-methionine:caffeic acid 3-O-methyltransferase - PAL phenylalanine:ammonia-lyase - XOMT S-adenosyl-l-methionine:xanthotoxol O-methyltransferase     

Time courses for phytochrome-induced enzyme levels in phenylpropanoid metabolism (phenylalanine ammonia-lyase,naringenin-chalcone synthase) compared with time courses for phytochrome-mediated end-product accumulation (anthocyanin,quercetin)

Time course for changes in the levels of enzymes characteristic of general phenylpropanoid metabolism (phenylalanine ammonia-lyase, PAL; EC 4.3.1.5) and of the flavonoid-glycoside branch pathway (naringenin-chalcone synthase, CHS; EC 2.3.1.74) were measured in the cotyledons of mustard (Sinapis alba L.) seedlings and compared with the rates of accumulation of related end products (anthocyanin and quercetin). Induction of enzyme levels and of end-product accumulation was carried out with red and far-red (FR) light, operating via phytochrome. The data are compatible with the concept that the phytochrome-mediated appearance of enzymes such as PAL and CHS is indeed a prerequisite for the appearance of anthocyanins and flavonols. However, there is no close correlation between enzyme levels and the rates of synthesis of end products which could justify the identification of specific rate-limiting enzymes. Rather, the data indicate that there is a second phytochrome-dependent step, beyond enzyme induction, where the actual rate of flavonoid accumulation is determined. Anthocyanin and quercetin accumulation respond differently to light. However, the relative action of continuous FR, red light pulses and stored phytochrome signal is the same in both cases. This indicates that the mode of operation of phytochrome is the same in both cases. The two syntheses differ only in the degree of responsiveness towards phytochrome. The time course for changes in CHS levels in continuous FR, i.e. under conditions of phytochrome photosteady state, is similar to the time course for PAL levels whereas the time courses in darkness, following transfer from FR to darkness, are totally different. In the case of CHS, a transient rise is observed whereas, with PAL, an instantaneous drop in enzyme level occurs after transfer from FR to darkness. It is concluded that the stored phytochrome signal operates in darkness in the case of CHS but not in the case of PAL.Abbreviations c continuous - CHS naringenin-chalcone synthase (EC 2.3.1.74) - FR far-red light (3.5 W·m^-2) - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Pfr phytochrome (far-red absorbing) - Pr phytochrome (red absorbing) - R red light (6.8 W·m^-2) - RG9-light long-wavelength far-red light obtained with RG9 glass filter - [Pfr]/[Ptot], whereby - Ptot total phytochrome (Pr+Pfr)     

Expression of the <Emphasis Type="Italic">Apx</Emphasis> gene family during leaf senescence of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxygen-free radicals are thought to play an essential role in senescence. Therefore, the expression patterns of the small gene family encoding the H₂O₂ scavenging enzymes ascorbate peroxidase (APX; EC 1.11.1.11) were analyzed during senescence of Arabidopsis thaliana (L.) Heinh. Applying real-time RT-PCR, the mRNA levels were quantified for three cytosolic (APX1, APX2, APX6), two chloroplastic types (stromal sAPX, thylakoid tAPX), and three microsomal (APX3, APX4, APX5) isoforms identified in the genome of Arabidopsis. The genes of chloroplastic thylakoid-bound tAPX and the microsomal APX4 exhibit a strong age-related decrease of mRNA level in leaves derived from one rosette as well as in leaves derived from plants of different ages. In contrast to the tAPX, the mRNA of sAPX was only reduced in old leaves of old plants. The microsomal APX3 and APX5, and the cytosolic APX1, APX2, and APX6 did not show remarkable age-related changes in mRNA levels. The data show that expression of the individual APX genes is differentially regulated during senescence indicating possible functional specialization of respective isoenzymes. The hydrogen peroxide levels seem to be controlled very precisely in different cell compartments during plant development.     

Expression of arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide

The effects of ozone or sulfur dioxide on antioxidant enzymes were investigated in Arabidopsis thaliana. Plants were fumigated with 0.1–0.15 ppm ozone or sulfur dioxide up to about 1 week in an environment-controlled chamber. Both pollutants increased the activities of ascorbate peroxidase and guaiacol per-oxidase in leaves, but had little effect on the activities of superoxide dismutase, catalase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase. Ozone was more effective than sulfur dioxide in increasing the activities of the peroxidases. Ascorbate peroxidase activity increased 1.8-fold without a lag period during fumigation with 0.1 ppm ozone, while guaiacol peroxidase activity increased 4.4-fold with a 1-day lag. Expression of the APX1 gene encoding cytosolic ascorbate peroxidase was further investigated. Its protein levels in leaves exposed to 0.1 ppm ozone for 4 or 8 days were 1.5-fold higher than in controls. Both ozone and sulfur dioxide elevated APX1 mRNA levels in leaves at 4 and 7 days, whereas at 1 day only ozone was effective. The induction of APX1 mRNA levels by ozone (3.4- to 4.1-fold) was more prominent than that by sulfur dioxide (1.6-to 2.6-fold). The APX1 mRNA level increased by day and decreased by night. Exposure of plants to 0.1 ppm ozone enhanced the APX1 mRNA level within 3 h, which showed a diurnal rhythm similar to that of the control. These results demonstrate that near-ambient concentrations of ozone as well as similar concentrations of sulfur dioxide can induce APX1 gene expression in A. thaliana.Environmental Biology Division     

Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-min red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue, light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response, could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings.Abbreviations Norflurazon NF-4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-tolyl)-3 (2H) pyridazinone - PAL phenylalanine ammonia-lyase - phytochrome photoequilibrium P_fr/P_tot - P_fr far-red absorbing form of phytochrome - P_r red absorbing form of phytochrome - P_tot total phytochrome: P_r+P_fr     

Induction of early blight resistance in tomato by <Emphasis Type="Italic">Quercus infectoria</Emphasis> gall extract in association with accumulation of phenolics and defense-related enzymes

Treatment of tomato leaves with aqueous extract (0.5%) of the galls of Quercus infectoria significantly reduced infection from subsequent inoculation with Alternaria solani, the tomato early blight pathogen. When the leaves were challenge-inoculated with A. solani 3 d after application of Q. infectoria gall extract (QIGE), the percent defoliation decreased from 33.6 to 7.3. Two to three day pre-treatment with QIGE reduced the percent defoliation by 77 percent. The biochemical responses of tomato plants to QIGE were also studied. In tomato plants treated with QIGE, phenolic content increased rapidly, reached the maximum at 2 d after treatment. Phenylalanine ammonia-lyase (PAL) activity increased significantly from 1 d after treatment and the maximum enzyme activity was recorded 2 d after treatment at which period a 3-fold increase in PAL activity was observed when compared to the control. Peroxidase (PO) activity was also significantly increased 1 d after treatment and the maximum activity was reached 2 d after treatment. Peroxidase isozyme analysis indicated that PO-1 was increased dramatically in tomato leaves 1 d after treatment and maintained at the same level throughout the experimental period of 6 d. When tomato leaves were treated with QIGE, a two-fold increase in chitinase and β-1,3-glucanase activities was recorded 2 and 3 d respectively, after treatment. The enhanced activities of defense-related enzymes and elevated levels of phenolics in QIGE-treated tomato plants between 1 and 3 d after treatment suggest that these induced biochemical defenses may be involved in the suppression of early blight by QIGE.     

Hydrogen peroxide is required for abscisic acid-induced NH4+ accumulation in rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced NH₄⁺ accumulation in rice leaves was investigated. ABA treatment resulted in an accumulation of NH₄⁺ in rice leaves, which was preceded by a decrease in the activity of glutamine synthetase (GS) and an increase in the specific activities of protease and phenylalanine ammonia-lyase (PAL). GS, PAL, and protease seem to be the enzymes responsible for the accumulation of NH₄⁺ in ABA-treated rice leaves. Dimethylthiourea (DMTU), a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced accumulation of NH₄⁺ in rice leaves. Inhibitors of NADPH oxidase, diphenyleneiodonium chloride (DPI) and imidazole (IMD), and nitric oxide donor (N-tert-butyl-α-phenylnitrone, PBN), which have previously been shown to prevent ABA-induced increase in H₂O₂ contents in rice leaves, inhibited ABA-induced increase in the content of NH₄⁺. Similarly, the changes of enzymes responsible for NH₄⁺ accumulation induced by ABA were observed to be inhibited by DMTU, DPI, IMD, and PBN. Exogenous application of H₂O₂ was found to increase NH₄⁺ content, decrease GS activity, and increase protease and PAL-specific activities in rice leaves. Our results suggest that H₂O₂ is involved in ABA-induced NH₄⁺ accumulation in rice leaves.     

Root colonization of different hosts by the vesicular-arbuscular mycorrhizal fungus Glomus dimorphicum

Colonization of the roots of beans, alfalfa, onions, red clover, corn, and four barley cultivars (Bonanza, Klondike, Gateway 63, and Olli) by Glomus dimorphicum Boyetchko and Tewari, a vesicular-arbuscular mycorrhizal fungus isolated from a barley field in Alberta, Canada, was studied under greenhouse conditions. Infection levels were low in all four barley cultivars but were higher in beans, alfalfa, and onions and were highest in red clover and corn roots. The infection patterns of G. dimorphicum varied among all the hosts. Coiling of intracellular hyphae occurred in corn, alfalfa, and red clover roots. Appreciable numbers of intraradical vesicles were found only in red clover and bean roots, while arbuscules formed in all hosts except barley. It was concluded that the pattern of root colonization by G. dimorphicum is influenced by the host genome and that the fungal morphology in the roots is variable and, thus, not diagnostic for the mycorrhizal species.     

转GhB301基因烟草的防御酶活性及抗病相关基因表达分析

为了明确棉花ERF-B3亚族转录因子基因GhB301在烟草异位表达后(抗枯萎病中)的功能,该研究以过表达GhB301基因烟草和野生型烟草为材料,采用枯萎病菌孢子悬浮液接菌方法,分析病原菌侵染前后防御酶活性变化以及防卫相关基因的表达变化与抗病性的关系。结果显示:(1)棉花枯萎病菌处理15d后,2个转基因株系烟草叶片黄化程度与野生型相比较轻。(2)棉花枯萎病菌处理后,过表达GhB301转基因烟草和野生型烟草叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性较未接菌对照显著提高,并且酶活峰值出现均早于野生型材料;转基因材料叶片的POD、PAL、PPO活性均在处理3d后达到峰值,而野生型材料叶片的POD、PAL活性在处理5d后才达到峰值。(3)接种棉花枯萎病菌后活性氧相关基因、乙烯(ET)/茉莉酸(JA)途径相关基因、病程相关基因的表达量在转基因株系OE1和OE2中均受到明显影响。研究推测,GhB301在烟草中的异位表达激活了防卫相关基因的表达,提高了防御酶的活性,从而增强了烟草对枯萎病菌的抗性。