不同抗性蚕豆品种根系分泌物对枯萎病菌的化感作用及根系分泌物组分分析

蚕豆枯萎病是土传病害,其发生与蚕豆根系分泌物有密切关系。本文以3个枯萎病不同抗性蚕豆品种——‘89-147’(高抗)、‘8363’(中抗)和‘云豆324’(感病)为材料,通过水培试验收集根系分泌物,测定根系分泌物对镰刀菌孢子萌发和菌丝生长的影响,分析对枯萎病表现出不同抗性的蚕豆品种根系分泌物中糖、氨基酸和有机酸的含量,分离鉴定了根系分泌物中氨基酸和有机酸的组分。结果表明,抗病品种的根系分泌物抑制了尖孢镰刀菌的孢子萌发和菌丝生长,在加入5 mL中抗品种根系分泌物时,显著促进尖孢镰刀菌孢子萌发,但对菌丝生长无显著影响;而在加入1 mL感病品种根系分泌物时,就能显著促进尖孢镰刀菌孢子萌发和菌丝生长。不同抗性蚕豆品种根系分泌物中氨基酸总量和总糖含量随抗性的降低而升高,有机酸分泌总量则随蚕豆品种对枯萎病的抗性增加而升高。感病品种和中抗品种中检出15种氨基酸,而高抗品种中检出14种,组氨酸只存在于中抗品种中,脯氨酸仅在感病品种中检出,3个蚕豆品种根系分泌物中均未检出精氨酸。蚕豆根系分泌物中天门冬氨酸、谷氨酸、苯丙氨酸、酪氨酸和亮氨酸含量高,可能会促进枯萎病的发生,而蛋氨酸、赖氨酸和丝氨酸含量高可能抑制枯萎病发生。酒石酸仅在抗病品种中存在,根系分泌物中有机酸种类丰富,有助于提高蚕豆对枯萎病的抗性。蚕豆对枯萎病的抗性不同,根分泌物对镰刀菌孢子萌发和菌丝生长的影响也不同,而这种抗病性差异与蚕豆根系分泌物中糖、氨基酸、有机酸的含量和组分密切相关。     

蚕豆根系分泌物中氨基酸含量与枯萎病的关系

通过田间试验研究不同品种蚕豆枯萎病病情指数的差异,并通过水培试验鉴定蚕豆根系分泌物中氨基酸组分并测定氨基酸的含量,分析各氨基酸组分与蚕豆枯萎病病情指数的相关性。结果表明:根系分泌物中氨基酸总量随着蚕豆枯萎病抗性的降低而升高。感病品种和中抗品种中检出15种氨基酸,而抗病品种中检出14种,组氨酸只存在于中抗品种中,脯氨酸仅在感病品种中检测到,3个蚕豆品种根系分泌物中均未检出精氨酸。丝氨酸(Ser)、蛋氨酸(Met)和赖氨酸(Lys)与枯萎病病指呈负相关关系,以Ser的相关系数最高,其他13种氨基酸含量与蚕豆枯萎病的病情指数呈正相关。蚕豆根系分泌物中Ser、Met和Lys含量及Ser/Gly、Ser/Ala比值高能抑制枯萎病的发生与发展,而天门冬氨酸(Asp)、苏氨酸(Thr)、甘氨酸(Gly)、丙氨酸(Ala)、缬氨酸(Val)、酪氨酸(Tyr)、苯丙氨酸(Phe)含量高时能促进枯萎病的发生。不同蚕豆品种根系分泌的氨基酸含量与组分的差异是影响蚕豆对枯萎病抗性差异的重要原因之一。     

荧光假单胞杆菌RB-42 RB-89的趋化性与烟草根部定殖研究

烟草PGPR(PlantGrowth PromotingRhizobacteria)菌株RB 89、RB 42对烟草根部收集液有较强的趋化性。在稀释浓度为10-5时,RB 89、RB 42的趋化值依次为1 79、1 83。RB 42和RB 89对氨基酸趋化性差异显著,对酪氨酸、脯氨酸、甘氨酸、笨丙氨酸表现出较强的趋化性,对组氨酸、苏氨酸没有表现出趋化性;RB 42和RB 89菌株对有机酸的趋化性没有明显区别,只是RB 42对乳酸的趋化性表现得比对其它有机酸的强些;菌株对糖类物质的趋化性最弱。菌株RB 89、RB 42在烟株根表的定殖受灭菌、趋化剂影响较大,土壤灭菌、趋化剂处理可增加菌株在根表的定殖量。     

籽粒苋不同富钾基因型根系分泌物中有机酸和氨基酸的变化特点

采用溶液培养试验研究了籽粒苋(Amaranthus.spp.)不同富钾基因型在不同供钾水平条件下3个不同生长时期内根系分泌物中氨基酸和有机酸的种类及含量变化情况。结果表明,1)籽粒苋根系分泌物中氨基酸和有机酸含量随着供钾水平的升高而降低,且富钾基因型始终大于一般基因型。在低钾胁迫时,根系分泌物中有10种氨基酸和5种有机酸出现,而在供钾正常时则缺少谷氨酸、甘氨酸、苯丙氨酸、草酸和柠檬酸等;酪氨酸、-氨基丁酸和丝氨酸的含量约占氨基酸分泌总量的一半以上;苹果酸含量约占有机酸分泌总量的60%以上。2)籽粒苋生长50d时,一般基因型氨基酸和有机酸的分泌总量较生长40d时迅速降低,而富钾基因型降低速度则相对较为平稳。在3个生长期内,酪氨酸、-氨基丁酸和丝氨酸均为两类基因型根系分泌物中的主要氨基酸种类,苹果酸则是主要的有机酸类型,其在氨基酸和有机酸分泌总量中所占相对比例均随生长期的延长而升高。3)籽粒苋根系分泌物处理后的土壤速效钾含量均高于清水对照处理,富钾基因型在低钾胁迫时的根系分泌物对土壤钾的活化作用明显大于一般基因型。     

化肥有机肥配施对油菜营养生长期根系分泌物的影响

探讨油菜营养生长期根系分泌有机酸和氨基酸对不同施肥处理的响应,为油菜安全高效生产提供理论依据。盆栽试验设置5 个化肥有机肥不同配施比例,分析油菜根系和地上部的干重及其根系分泌的有机酸和氨基酸的种类和含量。研究结果表明,油菜根系分泌有机酸以丙二酸、乙酸和柠檬酸为主,其中丙二酸和乙酸受施肥影响最为显著,有机肥配施显著降低二者分泌量。与单施化肥相比,有机肥配施显著减少了油菜根系分泌有机酸总量,并不同程度降低了地上部和根系生物量。化肥有机肥配施比例为1∶3 时有机酸总量降低最明显,但生物量与单施化肥无明显差异。油菜根系分泌氨基酸以谷氨酸和γ- 氨基丁酸为主,不同配施比例对油菜根系分泌35 种氨基酸种类的影响不大,但对氨基酸分泌总量影响显著。随有机肥配比的增加,氨基酸总量呈现先减少后增加的趋势。根系干重与有机酸分泌总量呈极显著正相关关系,但与氨基酸分泌总量无关,后者与土壤可溶性有机碳含量和细菌数量显著相关。油菜根系分泌有机酸和氨基酸所起的生态作用可能不同,前者可能是植物受到养分胁迫时的应激反应,后者可能是为了增强土壤活性。化肥有机肥1∶3 配比为油菜生长提供最均衡养分,为油菜施肥管理提供参考。     

荧光假单胞杆菌RB-42 RB-89的趋化性与烟草根部定殖研究

烟草PGPR(Plant Growth-Promoting Rhizobacteria)菌株RB-89、RB-42对烟草根部收集液有较强的趋化性。在稀释浓度为10^-5时，RB-89、RB-42的趋化值依次为1．79、1．83。RB-42和RB-89对氨基酸趋化性差异显著，对酪氨酸、脯氨酸、甘氨酸、笨丙氨酸表现出较强的趋化性，对组氨酸、苏氨酸没有表现出趋化性；RB-42和RB-89菌株对有机酸的趋化性没有明显区别，只是RB-42对乳酸的趋化性表现得比对其它有机酸的强些；菌株对糖类物质的趋化性最弱。菌株RB-89、RB一42在烟株根表的定殖受灭菌、趋化剂影响较大，土壤灭菌、趋化剂处理可增加菌株在根表的定殖量。     

大豆根系分泌物中氨基酸对根腐病菌生长的影响

采用砂培和室内模拟方法,研究了两个抗病性不同的大豆品种水溶性根系分泌物中氨基酸组分随作物生长的变化;同时检测了培养基中添加大豆根系分泌物和纯品氨基酸对大豆根腐病菌菌落生长的影响。结果表明,添加大豆苗期和花荚期根分泌物均显著促进尖镰孢菌菌丝生长,添加成熟期根分泌物显著促进腐皮镰孢菌菌丝生长。易感根腐病大豆品种合丰25号花荚期以后根分泌物中氨基酸种类多于抗根腐病大豆品种绥农10号。感病大豆品种根系分泌的氨基酸总量随生育时期增加,在鼓粒期达到最高;抗病大豆品种根系分泌的氨基酸总量在花荚期最高。感病大豆品种根系分泌的主要氨基酸为精氨酸,抗病大豆品种根系分泌的氨基酸主要为天冬氨酸。氨基酸纯品培养中,添加精氨酸和酪氨酸处理的尖镰孢菌菌落直径显著高于不加氨基酸的对照菌落直径;添加丝氨酸和天冬氨酸的处理菌落直径则显著低于对照处理。同时,添加天冬氨酸的培养基上腐皮镰孢菌菌落直径显著低于不加氨基酸的对照。可见,不同大豆品种根系分泌物中氨基酸组分对病原菌生长起着一定的作用,其表现的作用受根际氨基酸种类和氨基酸浓度影响较大,对于不同病原菌的作用存在差异。     

根系分泌物与重金属的化学行为研究

通过毛细管电泳测定根系分泌物的组成发现,根系在受到重金属铅、镉或铅-镉共同作用下分泌物种类与对照都有不同程度的变化;透析袋平衡透析方法则证实了根系分泌物与重金属之间存在着络合反应,络合能力的大小与重金属本身的性质有关,小麦根系分泌物对铅的络合能力大于镉。溶解试验表明,小麦分泌物对红壤中铅、镉的溶出没有显著的影响。小麦无菌根系分泌物、非无菌分泌物、非无菌过0.45m分泌物均促进红壤对铅、镉的吸附。其中铅吸附量较对照分别增加了12%,16%,21%;镉吸附量较对照分别增加了66%,102%,72%。     

油菜根系分泌物的GC-MS检测方法研究

为筛选建立BP-5MS毛细管柱检测油菜根系分泌物的方法,本文采用水培试验,利用CH2Cl2提取油菜幼苗的根系分泌物,通过GC-MS检测分离鉴定。结果表明,根据待测物质组分性质,采用合适的溶剂延迟,分阶段细化升温程序,检测出来的特征峰较多且分布密集,特征峰分离相对比较好。从检测结果看,油菜苗期根系分泌物主要包括烃类、醇类、酯类和酸类化合物,且烃类和醇类化合物的相对含量较高,这些物质大多数具有化感作用,对植物在逆境环境中的生长和发育有一定的影响。     

三种氨基酸对菜心产量和品质的影响

【目的】氨基酸能被作物吸收利用,影响作物的养分吸收和品质形成。采用盆栽试验, 通过三种氨基酸组氨酸（His）、 甘氨酸（Gly）和甲硫氨酸（Met）喷施,研究三种氨基酸对菜心生长及品质的影响。【方法】试验设置六个浓度梯度为0、 50、 100、 200、 400、 800 mg/kg。氨基酸喷施时间为34叶期、 78叶期和抽苔期。喷施5天后测定株高、 倒4叶叶长、 叶宽等生长指标。收获期地上部测重,测定Vc含量、 可溶性糖含量、 蛋白质含量,离子色谱法测定硝酸盐和草酸含量。【结果】三种氨基酸对菜心生长、 产量、 地上部蛋白质、 硝酸盐和草酸的影响如下, 1） 50~400 mg/kg的组氨酸和甲硫氨酸有增产效果,其中喷施100 mg/kg甲硫氨酸菜心产量增幅最大,达17.3%,氨基酸高浓度（800 mg/kg）喷施时,产量下降。2）三种氨基酸处理的菜心地上部蛋白质含量明显升高,且氨基酸喷施浓度和蛋白质含量之间呈极显著正相关关系（r=0.4026）。3）菜心地上部硝酸盐含量随氨基酸喷施浓度的提高先下降后上升,其中喷施100 mg/kg的甘氨酸对降低菜心地上部硝酸盐含量的效果最显著,比对照降低30.7%; 喷施高浓度氨基酸（800 mg/kg）其硝酸盐含量反而比对照高。4）草酸在植物体中普遍存在会影响蔬菜的品质,喷施氨基酸后菜心地上部草酸含量均下降,甲硫氨酸处理与对照相比可降低54.5%~68.3%。【结论】50~200 mg/kg的组氨酸、 甘氨酸和甲硫氨酸喷施能在不同程度上促进菜心生长和增产,提高菜心地上部蛋白质含量,降低硝酸盐和草酸含量。该研究结果可为氨基酸在蔬菜生产上的应用,尤其是如何降低草酸含量、 提高蔬菜品质提供依据。     

Influence of glyphosate on amino acid composition of Egyptian broomrape

The parasitic plant broomrape is entirely dependent on its host for reduced carbon and nitrogen and is also susceptible to inhibition by glyphosate that is translocated to the parasite through a host. Studies were conducted to examine the effect of broomrape parasitism on amino acid concentrations of two hosts: common vetch that is tolerant of low levels of glyphosate and oilseed rape that has been genetically engineered for glyphosate resistance. The influence of glyphosate on the amino acid content of broomrape and the two hosts was also examined. Amino acid concentrations in leaves and roots of parasitized common vetch plants were generally similar to those of the corresponding tissues of nonparasitized plants. Amino acid concentrations in broomrape were lower than those of the parasitized common vetch root. For common vetch, glyphosate applied at rates that selectively inhibited broomrape growth did not alter individual amino acid concentrations in the leaves, but generally increased amino acid levels at 0.18 kg ha-1. Glyphosate application also increased the amino acid concentrations, with the exception of arginine, of broomrape growing on common vetch and did not generally influence concentrations in leaves or roots of common vetch. In oilseed rape, parasitization by broomrape generally led to higher amino acid concentrations in leaves but lower concentrations in roots of parasitized plants. Broomrape had higher amino acid concentrations than roots of the parasitized oilseed rape. Glyphosate applied at 0.25 and 0.5 kg ha-1 generally increased the amino acid concentrations in oilseed rape leaves, but the 0.75 kg ha-1 application caused the amino acid concentrations to decrease compared to those of untreated plants. In oilseed rape root the general trend was an increase in the concentration of amino acids at the two highest rates of glyphosate. Individual amino acid concentrations in broomrape attachments growing on oilseed rape were generally increased following glyphosate application of 0.25 kg ha-1. These results indicate that low rates of glyphosate alter amino acid profiles in both host and broomrape and raise questions about the regulation of amino acid metabolism in the parasite.     

Effect of cadmium on the chemical composition of xylem exudate from oilseed rape plants (Brassica napus L.)

Abstract

Phytoremediation is a good technique for removing cadmium (Cd) from farmland soils. To remove Cd from these soils effectively, it is necessary for Cd ions to be transported to the shoot organs for later harvest. However, the mechanism of Cd translocation to shoot organs via xylem vessels has not yet been elucidated. We selected oilseed rape plants (Brassica napus L.) and established a method to collect xylem exudates from these plants. After 3 days of Cd treatment (10 µmol L^?1 and 30 µmol L^?1) the Cd concentrations in the xylem exudates were approximately 6.5 µmol L^?1 and 16 µmol L^?1, respectively. The detection of Cd in the xylem exudate indicated that Cd was moving to shoot organs via xylem vessels. The effect of these Cd treatments on the amino acid, organic acid and protein composition of xylem exudates from oilseed rape plants was investigated. The level of amino acids and organic acids detected was enough to bind Cd transported via the xylem. Sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis revealed that proteins with molecular weights of 36 kDa and 45 kDa clearly increased in the exudates with Cd treatment. The possibility that these compounds are binding Cd in the xylem exudates was discussed.     

果胶细菌诱导油菜结瘤与固氮研究

果胶细菌处理油菜幼根（2天苗龄、根长3cm），同时接种紫云英根瘤菌突变菌株（Rhizobiumhuakuii），诱发类根瘤的结瘤率为96.80％。从这些类根瘤中分离的菌株能回接到原寄主。类根瘤的电镜观察可以明显地见到细胞内充满了细菌。乙炔还原法测定的固氨酶活性（C2H4）为76.80nmol／（planth）。     

STUDIES ON DIFFERENCES OF NITROGEN EFFICIENCY AND ROOT CHARACTERISTICS OF OILSEED RAPE (BRASSICA NAPUS L.) CULTIVARS IN RELATION TO NITROGEN FERTILIZATION

Differences of nitrogen efficiency of oilseed rape (Brassica napus L.) cultivars and their physiological properties were studied in a pot experiment, and the ratio of seed yield with no nitrogen supplied to that with normal nitrogen supply was adopted as a nitrogen efficiency coefficient. Results showed that the nitrogen efficiency coefficient determined for eight oilseed rape cultivars varied from 0.37 to 0.69, the ratio of nitrogen uptake amounts per plant, nitrogen transfer velocity from stems and leaves to seeds, and nitrogen physiological efficiency of oilseed rape cultivars under nitrogen stressed condition differed from with normal nitrogen supply. The higher the nitrogen efficiency of a cultivar, the higher the ratio of N uptake in no nitrogen to with N supplied. Under low nitrogen-supplying conditions, high nitrogen efficiency cultivars had longer roots, more lateral roots, higher amounts of reuse of nitrate from stem and leaves, and higher nitrate reductase activities in leaves.     

Amino acids exuded from axenic roots of lettuce and white lupin seedlings exposed to different cadmium concentrations

The objective of this study was to evaluate if amino acids in roots and/or in root exudates play a role in cadmium (Cd) stress. Lettuce (Lactuca sativa L. cv. Reine de Mai) and white lupin (Lupinus albus L. cv. lublanc) were grown for 19 to 21 days with axenic roots in a hydroponic system. After treatment with various concentrations of Cd (0, 0.01, 0.1, 1, 10, and 100 μM Cd) per nine days, roots and root exudates were collected. The stress did not result in significant dry weight (DW) differences between Cd‐treated and control plants, but Cd induced decreases in relative water content (RWC) and water potential (Pm). Amino acid levels and carbon (¹⁴C) incorporation into amino acids increased at low Cd concentrations in roots. However, 100 μM Cd induced a decrease of amino acid levels and an equally significant reduction of ¹⁴C incorporation, suggesting a decreased plant metabolism. Moreover, a higher Cd concentration induced increased levels of specific amino acids, for instance asparagine and lysine in lettuce and asparagine and hydroxylysine in lupin roots. Amino acids in root exudates corresponded less than 1% of the amounts found in root cells suggesting that amino acids could not be the major Cd chelators. Amino acid accumulation in root exudates differed than that found in roots except for asparagine. In conclusion, Cd induces in the root and root exudates increased levels of specific amino acids, such as Asn, Lys and HLys similarly to other environmental stresses. Although the amino acids could not participate in Cd chelation, lysine and its derivatives, such as hydroxylysine, could be used as stress markers for Cd in higher plants.     

Analysis of amino acid uptake and translocation in Arabidopsis with a low‐cost hydroponic system

In soils, amino acids may be an important source of nitrogen for plants, at least in those where organic matter is not quickly degraded. The physiology of uptake of amino acids by roots was mainly studied in the 70's and 80's, before genes encoding amino acid importers were cloned in the 90's. While two families of amino acid transporters have been identified, yielding a total of about 100 genes, the role of each member is yet to be elucidated. As a tool for studying the role of amino acid transporters from Arabidopsis we set up a new hydroponic system suitable for radioisotope use. This system enables reproducible amino acid uptake by roots and estimation of the transport to the shoots of the amino acid taken up. We show that the rates of glutamine (Gln) uptake by wild‐type roots and transfer to the shoots were linear, and that other tested amino acids were translocated to the shoots with lower efficiency than Gln. A T‐DNA insertion mutant for a Gln exporter was compared to the wild‐type plants. Gln uptake and transfer were similar in both genotypes, showing that the suppression of the exporter did not affect uptake or transfer of amino acids to the shoots. The main advantage of the hydroponic system presented here is that all the materials used to grow Arabidopsis are virtually free and can therefore be discarded, a useful feature when working with radioactivity.     

Extraktion eines kupferbelasteten Bodens unter Verwendung eines aminosäurehaltigen Reststoffhydrolysats. 2. Elutionsdynamik und Bilanzierung der Aminosäuren nach kontinuierlichem Eintrag in Bodensäulen

Extraction of a copper contaminated soil material by the percolation of an amino acid containing residue hydrolysate. 2. Time course of amino acid elution and input/output balance of amino acids During 16 days an amino acid containing blood meal hydrolysate (amino acid concentration: 188 mMol·L^?1) was percolated through a column packed soil material (soil content per column: 4.1 kg dry weight, four parallels). The copper contaminated material (soil type: Typic Udifluvent, soil texture: sandy loam, loamy sand) was sampled from an area formerly used for cultivation of hop (Humulus lupus). Besides the investigation of the copper liberation the experiments aimed to determine the elution dynamic and input/output balance of amino acids (time span for amino acids balance 14 days). In total 11.7 L of hydrolysate, containing 2.2 Mol of amino acids, were introduced into each column. The mean amino acid output with the column effluent was 1.13 Mol. This corresponds to an elution degree of 51.2%, related to the sum of applicated amino acids, and to a mean substance specific elution degree of 48.4% reflecting the elution of 15 compounds. The substance specific elution ranged from 9.6% (serine) to 75.5% (valine). The highest concentrations of serine and threonine were determined in the effluents after two days, whereas the histidine concentration was highest at the last sampling. The differences in the percolation properties of the amino acids are discussed in terms of important retention and elimination processes (biodegradation, ad-/desorption, intercalation).     

Impact of fresh root material and mature crop residues of oilseed rape (Brassica napus) on microbial communities associated with subsequent oilseed rape

Glasshouse bioassays were conducted to assess the impact of different inputs of oilseed rape plant material on soil and rhizosphere microbial diversity associated with subsequently grown oilseed rape (Brassica napus) plants. The first bioassay focussed on the effect of oilseed rape rhizodeposits and fresh detached root material on microbial communities, in a rapid-cycling experiment in which oilseed rape plants were grown successively in pots of field soil for 4 weeks at a time, with six cycles of repeated vegetative planting in the same pot. Molecular analyses of the microbial communities after each cycle showed that the obligate parasite Olpidium brassicae infected the roots of oilseed rape within 4 weeks after the first planting (irrespective of the influence of rhizodeposits alone or in the presence of fresh detached root material), and consistently dominated the rhizosphere fungal community, ranging in relative abundance from 43 to 88 % when oilseed rape was grown more than once in the same soil. Fresh detached root material also led to a reduction in diversity within the soil fungal community, due to the increased relative abundance of O. brassicae. In addition, rhizosphere bacterial communities were found to have a reduced diversity over time when fresh root material was retained in the soil. In the second glasshouse experiment, the effect of incorporating mature, field-derived oilseed rape crop residues (shoots and root material) on microbial communities associated with subsequently grown oilseed rape was investigated. As before, molecular analyses revealed that O. brassicae dominated the rhizosphere fungal community, despite not being prevalent in either the residue material or soil fungal communities.     

不同品种油菜氮效率差异及其成因分析

采用盆栽试验，以氮胁迫与正常供氮条件下籽粒产量的比值作为氮效率系数，探讨了不同品种油菜氮效率差异及其生理基础。结果表明，所测定的8个油菜品种氮效率系数的变化范围是0．37～0．69；氮胁迫与正常供氮条件下不同油菜品种植株氮素累积吸收量、生长后期茎叶氮素转运率及氮素生理效率的比值不同，氮效率高的品种，其比值高，反之亦然;在氮素供应水平低的情况下，氮效率高的品种具有较长的根长、较多的侧根、较高的茎叶硝态氮再利用量和叶片硝酸还原酶活性。     

Precolumn phenylisothiocyanate derivatization and liquid chromatography of amino acids in food

A precolumn phenylisothiocyanate derivatization method is described for the determination of amino acids in protein hydrolysates from a wide variety of complex food matrixes, with and without performic acid oxidation pretreatment. Analysis of samples that were not pretreated with performic acid was necessary since this pretreatment destroyed an average of 25% of the histidine and 87% of the tyrosine present in the food samples. This method is rapid and reproducible; coefficients of variation between duplicate analyses of the same food item were less than 5% for a majority of the amino acids. Occasionally, variation between duplicate analyses for histidine and tyrosine was greater than 10%. Recoveries of amino acids added to samples were in the 100% range.