Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.     

Nonprotein amino acid composition of flatpea (Lathyrus sylvestris L.) as affected by ethephon seed treatments and seedling fertilization

Use of flatpea (Lathyrus sylvestris L.) as a forage is limited because of nonuniform seed germination and the potentially toxic effects of 2,4-diaminobutyric acid (A₂bu), a nonprotein amino acid found in seeds and vegetative tissues. The effects of ethephon (2-chloroethyl phosphonic acid) on seed germination, amino acid leachates of seeds, and amino acid composition (particularly A₂bu) of seedlings were investigated. Germination of flatpea seeds, imbibed for 16 h in 0, 100, 200, 400, 800, and 1600 mg/L ethephon, did not differ, but amino acid leachates tended to increase up to 200 mg/L ethephon and then decline at higher concentrations. The major amino acid constituents in leachates were A₂bu, 4-aminobutyric acid (Abu), and homoserine (Hse). Dry matter accumulation of seedlings grown from ethephon-treated seeds was reduced for second cuttings grown from ethephon-treated seeds and high nitrogen grown plants. During regrowth, free amino acid accumulation was most pronounced in leaves of plants supplied with high nitrogen. The most abundant free amino acids in flatpea tissues were the same as those in seed leachates, but concentration and relative abundance varied with nitrogen level, plant part, and ethephon treatment. Results suggest that ethephon seed treatments can have persistent effects on the growth and amino acid composition of flatpea seedlings grown under different nitrogen regimes.     

Nonprotein amino acid composition of flatpea (Lathyrus sylvestris L.) as affected by ethephon seed treatments and seedling fertilization

Use of flatpea (Lathyrus sylvestris L.) as a forage is limited because of nonuniform seed germination and the potentially toxic effects of 2,4-diaminobutyric acid (A₂bu), a nonprotein amino acid found in seeds and vegetative tissues. The effects of ethephon (2-chloroethyl phosphonic acid) on seed germination, amino acid leachates of seeds, and amino acid composition (particularly A₂bu) of seedlings were investigated. Germination of flatpea seeds, imbibed for 16 h in 0, 100, 200, 400, 800, and 1600 mg/L ethephon, did not differ, but amino acid leachates tended to increase up to 200 mg/L ethephon and then decline at higher concentrations. The major amino acid constituents in leachates were A₂bu, 4-aminobutyric acid (Abu), and homoserine (Hse). Dry matter accumulation of seedlings grown from ethephon-treated seeds was reduced for second cuttings grown from ethephon-treated seeds and high nitrogen grown plants. During regrowth, free amino acid accumulation was most pronounced in leaves of plants supplied with high nitrogen. The most abundant free amino acids in flatpea tissues were the same as those in seed leachates, but concentration and relative abundance varied with nitrogen level, plant part, and ethephon treatment. Results suggest that ethephon seed treatments can have persistent effects on the growth and amino acid composition of flatpea seedlings grown under different nitrogen regimes.     

Composition and Distribution of Free Amino Acids in Flatpea (Lathyrus sylvestris L.) as Influenced by Water Deficit and Plant Age

Drought-stressed flatpea (Lathyrus sylvestris L.) plants from8 to 22 weeks old were analysed for nitrogen, soluble proteinand free amino acids. An increase in nitrogen and free aminoacid concentrations and a decrease in soluble protein levelwere observed in roots of plants up to 16 weeks old. The cumulativeconcentration of free amino acids increased with drought stress.Tissue concentrations of 2, 4-diaminobutyric acid (1.6–2.6%of the dry weight) were highest in leaves. Levels increasedsteadily, nearly doubling, in leaves and stems between weeks10 and 16. Levels in drought-stressed leaves were, on average,11.9% higher than those of controls. Estimated concentrationsof a mixture of 4-aminobutyric acid and an unknown amino acidwere highest in stems, increased in this tissue with age andtended to increase in stems and leaves and decrease in rootsin response to water deficit. Levels of the mixture of homoserineand another unidentified amino acid were not influenced by ageor water status of the plants. Root concentrations of asparagine,arginine, glutamine, aspartate, and another prominent, unidentifiedamino acid increased with plant age and reached a peak at thetime of flowering (14 to 18 weeks). Only the concentration ofthe unknown compound was elevated following drought stress.Concentrations of valine, isoleucine, leucine, phenylalanine,and methionine also increased during this period and were elevatedin drought-stressed plants. Proline levels increased with plantage and drought stress, but proline accounted for only about10% of the total free amino acids in the drought-stressed plants. Key words: 2, 4-Diaminobutyric acid, drought, flatpea     

Calystegine distribution in potato (Solanum tuberosum) tubers and plants

Potato plants contain calystegines in leaves, stems, flowers, fruits and roots. Calystegines A₃ and B₂ are the main constituents. Highest concentrations were measured in sprouts emerging from the tubers. In 3 mm long sprouts, 3.3 mg total calystegines per g fresh mass were detected. Dormant tubers directly after harvest contain less calystegines in all parts than sprouting tubers. Flowers and young leaves are the aerial plant tissues with the highest calystegine concentration, i.e. 150 μg total calystegines per g fresh mass. Calystegine levels did not rise when sprouts were wounded. Tropinone application to sprouts and aerial tissues lead to an accumulation of pseudotropine and not to tropine. That indicates that stereospecific tropinone reduction is active in potato.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Identification of nickel chelators in three hyperaccumulating plants: an X-ray spectroscopic study

We have investigated the accumulation of nickel in a hyperaccumulating plant from the Brassicacae family Leptoplax emarginata (Boiss.) O.E. Schulz. Two supplementary hyperaccumulating plants, which have been the subject of a high number of publications, Alyssum murale Waldst. & Kit and Thlaspi caerulescens J.&C. Presl, and a nonaccumulating species Aurinia saxatilis were also studied for reference. The plants were grown during 4 months in specific rhizoboxes with Ni-bearing minerals as a source of nickel. Nickel speciation was analyzed through X-ray absorption spectroscopy at Ni K-edge (X-ray absorption near edge spectroscopy and extended X-ray absorption fine structure spectroscopy) in the different parts of the plants (leaves, stems and roots) and compared with aqueous solutions containing different organo-Ni(II) complexes. Carboxylic acids (citrate, malate) appeared as the main ligands responsible of nickel transfer within those plants. Citrate was found as the predominant ligand for Ni in stems of Leptoplax and Alyssum, whereas in leaves of the three plants, malate appeared as the chelating organic acid of accumulated metal. Histidine could not be detected either in leaves, stems nor roots of any studied plant sample.     

林生山黧豆中2,4—二氨基丁酸合成代谢的体外酶学研究

用~3H-天门冬氨酸为底物,林生山黧豆(Lathyrus sylvestris L.)叶片匀浆上清液为粗酶液,进行体外反应。结果表明,天门冬氨酸的放射性掺入到2,4-二氨基丁酸,加入谷氨酸则能抑制这种掺入。将上述粗酶液透析,加入可能的辅助因子,天门冬氨酸的放射性也掺入到2,4-二氨基丁酸。研究证实在体外天门冬氨酸可以作为2,4-二基丁酸合成的底物,在林生山黧豆体内存在催化天门冬氨酸转变为2,4-二氨基丁酸的合成酶(系)。以2,4-二氨基丁酸和γ-氨基丁酸为底物,用氨基酸自动分析仪测定产物含量,结果表明,2,4-二氨基丁酸和γ-氨基丁酸不互相转变。     

Distribution and transport of IAA in tomato plants

We determined as to indole-3-acetic acid (IAA), endogenous levels byliquid chromatography-mass spectrometry using ¹³C₆-IAA, diffusible levels byfluorometric detection using indole-propionic acid, and polar transportactivityby radioactive IAA in 3-month-old tomato plants (stems, leaves or roots). TheIAA concentration in the apoplast (AP) solution was higher than those in thesymplast (SP) solution in both the upper and lower parts of stems, showing thatIAA analysis of AP solution is important. Younger leaves exported much morediffusible IAA than older leaves. The IAA concentration in the main roots wasalmost at the same level as in the stems. The results suggested that thetransport capacity of IAA is probably the limiting factor for the amount of IAAtransported in stems and the amount of polar IAA transport might be only 19% ofthe endogenous IAA amount in stems.     

Variation in the nutritional value of leaf and stem fractions of nineteen leucaena lines

In two experiments, 2.5-month-old edible regrowth of the genus Leucaena (varying in tolerance to the leucaena psyllid pest attack) were analysed for nutritive value. In Experiment 1, forage of nine lines was separated into young leaves, young stems (within woody fraction < 5 mm diameter), old leaves and old stems (within a woody fraction 5-10 mm diameter) whereas in Experiment 2, forage of the other 10 lines was separated in main stem leaves, branch leaves, young branches, young main stems, old main stems and old branches for analysis. Major differences were recorded between lines and forage fractions. Nutrient contents in dry matter were: nitrogen 0.7-4.8%, phosphorus 0.1-0.3%, potassium 0.2-2.5%, calcium 0.2-1.6%, magnesium 0.1-0.6%, acid detergent fibre 20-63%, lignin 8-23% and in vitro dry matter digestibility 15-69%. The leaves and immature fractions were higher in nitrogen, phosphorus, potassium and digestibility but lower in calcium, magnesium and fibre. Nutrient content in L. Leucocephala lines exceeded that in L. esculenta, L. diversifolia, leucaena hybrids and the composites. Based on the data, a management strategy that will promote young growth is suggested as appropriate for high quality forage production.     

外源硫化氢对盐碱胁迫下裸燕麦叶片有机酸和激素含量的影响

为探讨外源硫化氢(H₂S)对盐碱胁迫下植物有机酸和激素水平的调控效应,以裸燕麦(Avena nuda)为材料,研究喷施50μmol·L^-1H₂S供体硫氢化钠(NaHS)溶液对3.00 g·kg^-1盐碱胁迫下叶片有机酸、激素含量和产量性状的影响。结果表明：盐碱胁迫显著提高了琥珀酸、丁烯二酸、苹果酸、葡萄糖醛酸和总有机酸含量,显著降低了焦谷氨酸、茉莉酸-异亮氨酸(JA-Ile)、反式-玉米素(tZ)和N⁶-(Δ²-异戊烯)腺嘌呤(iP)含量。喷施NaHS溶液显著提高盐碱胁迫下裸燕麦叶片中3-羟基-3-甲基谷氨酸、吲哚乙酸(IAA)、赤霉素A₇(GA₇)、茉莉酸甲酯(MJA)、iP含量和IAA/ABA比值,显著降低葡萄糖醛酸、赤霉素A₃(GA₃)、赤霉素A₄(GA₄)、总赤霉素(GA_S)、1-氨基环丙烷羧酸(A...     

Salicylic acid-induced adaptive response to copper stress in sunflower (Helianthus annuus L.)

The ameliorative effect of salicylic acid (SA: 0.5 mM) on sunflower (Helianthus annuus L.) under Cu stress (5 mg l⁻¹) was studied. Excess Cu reduced the fresh and dry weights of different organs (roots, stems and leaves) and photosynthetic pigments (chlorophyll a, b and carotenoids) in four-week-old plants. There was a considerable increase in Chl a/b ratio and lipid peroxidation in both the roots and leaves of plants under excess Cu. Soluble sugars and free amino acids in the roots also decreased under Cu stress. However, soluble sugars in the leaves, free amino acids in the stems and leaves, and proline content in all plant organs increased in response to Cu toxicity. Salicylic acid (SA) significantly reduced the Chl a/b ratio and the level of lipid peroxidation in Cu-stressed plants. Under excess Cu, a higher accumulation of soluble sugars, soluble proteins and free amino acids including proline occurred in plants treated with 0.5 mM SA. Exogenous application of SA appeared to induce an adaptive response to Cu toxicity including the accumulation of organic solutes leading to protective reactions to the photosynthetic pigments and a reduction in membrane damage in sunflower.     

A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates

This paper suggests a simple modification of the Ellman procedure when used to measure accurate changes in sulfhydryl (-SH) content induced by reactive oxygen intermediates (ROI). This modification became necessary when we found that the standard technique did not produce time invariant results in the presence of ROI-generating systems. Cysteine (cys; 20–100 μM) in 20 mM imidazole buffer (pH 7.0) containing 1.0 mM EDTA was reacted with excess (0.2 mM) 5,5′-dithiobis(2-nitrobenzoic acid), DTNB. The absorbance of the product (p-nitrothiophenol anion) was recorded at 412 nm (A₄₁₂). This A₄₁₂ was stable for 60 min and gave a linear relationship with cys concentrations used. ROI were generated either by 0.01 U xanthine oxidase (XO) + 0.01–1.0 mM hypoxanthine (HX), 0.01–1.0 mM H₂O₂, or H₂O₂ + 100 μM FeSO₄. In the presence of ROI, A₄₁₂ decreased with time and its rate of decrease was dependent upon the concentration of components of the ROI-generating system. This time-dependent decrease in A₄₁₂ was prevented completely by the addition of 100 U of catalase (CAT). Therefore, we modified the DTNB method as follows: -SH groups were reacted with ROI for 30 min; this was followed by the addition of 100 U of CAT to scavenge the excess unreacted ROI before the addition of DTNB to generate the product. Using this modification the ROI-induced decrease in A₄₁₂ was stable with time and was linearly related to the cys concentration. We further tested the modified procedure using metallothionein (MT) as a substrate for the ROI-induced changes in -SH content. MT, at concentrations of 2.5, 5.0, and 7.5 μM, was treated with XO + 100 μM HX. Using the modified procedure, an average decrease (as compared to the untreated control) of 15, 22, and 33 μM in -SH content was observed consistently at the respective MT concentrations. However, without the modification in the procedure, these average decrease were 20, 38, and 51 μM, respectively and continued to further increase with time. These discrepancies could give rise to errors ranging from 28 to 35% or higher in determination of the ROI-induced decrease in the -SH groups of MT. This data suggests that scavenging the unreacted H₂O₂ with C prior to the addition of DTNB to the assay mixture gives a stable and accurate estimate of the ROI-induced oxidative damage to -SH groups.     

氮磷添加对巨桉幼苗生物量分配和C:N:P化学计量特征的影响

巨桉(Eucalyptus grandis)是一种优良的速生用材树种, 了解氮(N)和磷(P)对巨桉生长、养分限制、化学计量特征的影响对于科学合理施肥具有重要意义。该实验以巨桉无性系组培苗为研究对象, 通过在酸性紫色土中设置不同施N或施P梯度, 研究巨桉幼苗各器官(根、茎、叶)生物量及碳(C)、N、P的分配和化学计量特征以及巨桉生长的养分限制状况。结果表明: 施N处理对巨桉根茎叶及总生物量的影响极显著, 增加了地上部分的生物量比例而显著降低了根系的生物量比例; 施P对巨桉幼苗总生物量影响不显著, 但显著提高了根的生物量分配比例, 对茎和叶的生物量分配没有显著影响。施N或施P显著改变了巨桉幼苗的N、P含量和化学计量比, 同时也显著影响了土壤与植物N:P的关系。施N可以促使酸性紫色土条件下巨桉对N的吸收而抑制对P的吸收, 施P则促进巨桉幼苗对P的吸收。施N对巨桉幼苗根茎叶的C、N、P分配特征有极显著影响, 而施P对巨桉幼苗根茎叶的C、N、P分配没有显著影响。施N极显著降低了巨桉幼苗N的利用率, 显著提高了P的利用率, 而施P处理极显著降低了巨桉幼苗P的利用率。从巨桉生物量沿施肥梯度和N:P的变化规律可以判断, 当叶片N:P < 15时, 巨桉的生长主要受到N的限制作用。施N可以显著地提高根茎叶的N:P比值, 缓解巨桉缺N的现象, 施P则进一步加剧了N元素的缺乏。     

Gibberellin-like activity in pea seedlings during growth under red,blue and white light

The influence of blue, red and white light and gibberellic acid (GA₃) on gibberellin-like activity in tissue extracts of leaves, stems and roots was investigated during growth of pea seedlings (Pisum salivum L. cv. Bördi). Higher GA-like activity was found in leaves and stems of pea plants that were growing in blue light than in those under red or white light. Patterns of change of activity were different in leaves, stems and roots, and in GA₃-treated plants.     

Production of caffeic,chlorogenic and rosmarinic acids in plants and suspension cultures of Glechoma hederacea

Glechoma hederacea L. (Lamiaceae) is a perennial plant, which is distributed widely in Europe, Asia and America. Important anti-oxidant compounds are caffeic acid esters like rosmarinic acid (RA) and chlorogenic acid (CA). Phenylalanine ammonia-lyase (PAL) and rosmarinic acid synthase (RAS, 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) contribute to the formation of RA. Our aim in this study was to follow the accumulation of RA, CA and caffeic acid in a suspension culture of G. hederacea. Growth, medium and secondary metabolism parameters were determined during a culture period of 14 days. The maximal PAL activity was observed on day 5 and the maximal RAS activity on day 8. The RA content was exceedingly high and reached 25.9% of the dry mass on day 7. Caffeic acid and CA contents remained rather low. Furthermore, the presence of RA, CA and caffeic acid and the expression patterns of RAS and hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HST), an important enzyme of monolignol formation, in leaves, flowers, stems and roots of naturally grown G. hederacea were assessed. The expression of RAS and HST genes was detectable in all organs except roots. Flowers accumulated 12.5% RA in their dry mass, leaves, stems and roots about 1%. CA was highest in leaves (2.0%), while it was at 1.6% in flowers, 1.3% in stems and almost undetectable in roots. The caffeic acid content remained at or below 0.4% of the dry weight in all organs.     

Changes in alfalfa forage quality and stem carbohydrates induced by arbuscular mycorrhizal fungi and elevated atmospheric CO2

Alfalfa is a widely distributed forage legume whose leaves are high in protein content and whose stems are suitable for bioethanol production. However, alfalfa forage digestibility, quality and yield may vary under future climate change scenarios. This legume can establish double symbiosis with nitrogen‐fixing bacteria and arbuscular mycorrhizal fungi (AMF). The presence of AMF can modify the evolution of biomass production and partitioning during the vegetative growth of alfalfa. We hypothesised that mycorrhizal symbiosis may change the quantity and/or quality of carbohydrates and lignin in leaves and/or stems of alfalfa, with these changes being dependent on the atmospheric CO₂ concentration at which plants are grown. Results showed that mycorrhizal alfalfa plants exposed to elevated CO₂ had improved leaf, stem and root biomass, enhanced amount of hemicellulose and decreased concentration of lignin in cell walls of leaves as well as increased levels of glucose and fructose in stems compared with non‐mycorrhizal alfalfa. These results indicated improved forage quality (leaves) and enhanced potential for bioethanol conversion (stems) in mycorrhizal alfalfa cultivated under elevated CO₂. Moreover, the potential of stems for producing CH₄ reinforced their suitability for the conversion of biomass into bioethanol.     

Responses of phenolic acid and defensive enzyme activities to mechanical damage in Artemisia frigida

Aims The study aims at understanding the effects of feed intake and trample damage on the phenolic acid formation and antioxidant enzyme activities in Artemisia frigida, and elucidating the adaptive mechanisms in A. frigida to grazing in secondary metabolites and their related enzyme activities.
Methods We analyzed the phenolic acid content and the activities of polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) and protective enzymes in leaves and roots in A. frigida under three levels (light, moderate, and heavy) of manipulative grazing condition. The measurements of the 9 phenolic acid contents started after 6 h of the mechanical damage of the plants by using the high performance liquid chromatography (HPLC), and the enzyme activities in leaves and roots were measured by a spectrophotometry method.
Important findings The light damage treatment induced productions of PPO, PAL and significantly (p < 0.05) increased antioxidant enzyme activities in the leaves and roots of A. frigida. The contents of PPO, PAL and antioxidant enzymes increased with increasing intensity of mechanical damage. Compared to the control, the content of free caffeic, syringic, ferulic and cinnamic acid in the leaves A. frigida were significantly elevated (p < 0.05) by 150.4%, 93.5%, 154.4% and 121.7%, respectively. They were significantly (p < 0.05) positively correlated with PAL activity in the moderate damage treatment. The content of free chlorogenic acid and catechol decreased by 91.1%, and 69.3%, respectively, compared with the control they had a negative correlation with PPO activity in the heavy damage treatment. The contents of gallic and protocatechuic acids increased (p < 0.05) by 280.6% and 215.7%, respectively, in the heavy damage treatment. With increasing intensity of mechanical damage, the content of 9 free phenolic acids significantly increased in roots but the increasing range was less than the one in leaves. Mechanical damage induced an increasing trend in the total amount of free and bounded phenolic acids in the leaves but a decreasing trend in the total amount of bounded phenolic acids in the roots of A. frigida. The results indicated that mechanical damage could firstly induce an increase of antioxidant enzymes and key enzymes in phenolic metabolism in A. frigida, leading to the accumulation of antioxidant substances of phenolic acids, further regulate the biosynthesis of lignins, quinones and tannins, and then enhance the resistance to mechanical damage and improved the tolerance of A. frigida to grazing.     

茶树水通道蛋白基因的克隆与表达分析

从茶树中克隆了6个水通道蛋白(aquaporin,AQP)基因的cDNA全长序列,根据序列相似性和系统进化分析结果,分别命名为CsNIP1;1、CsNIP5;1、CsPIP2;1、CsPIP2;2、CsTIP1;1和CsTIP2;2。氨基酸序列特征分析表明,6个基因的编码氨基酸序列长度在250~301个氨基酸残基,分子量在25.186~30.728kD之间;均为疏水性蛋白,并都含有6个跨膜螺旋;6个基因均含有MIP蛋白家族的特征序列HF/I/VNPA/SI/L/VTI/FA/G和NPA(Asn-Pro-Ala)基序,以及类似沙漏状的跨膜三级结构。荧光定量PCR分析显示:这6个基因在根、茎、叶和花中均表达,且在根中的表达水平最高,表明它们与茶树根系的物质转运密切相关;CsAQPs基因的表达受ABA、高盐、干旱和低温胁迫的调控,表明它们可能参与茶树抗逆响应。