Effects of designed sulfhydryl groups and disulfide bonds into soybean proglycinin on its structural stability and heat-induced gelation

The gel-forming ability of glycinin is one of soybean's most important functional properties. The proglycinin A1aB1b homotrimer was engineered to introduce sulfhydryl groups and disulfide bonds, and their effects on the structural stability and the heat-induced gelation were evaluated. On the basis of the crystal structure, five mutants were designed and prepared: R161C and F163C forming an interprotomer disulfide bond with the inherent free cysteine residue of Cys377, N116C/P248C forming a new intraprotomer disulfide bond, and N116C and P248C introducing a new sulfhydryl group. Mutants of R161C, F163C, and N116C/P248C formed a new disulfide bond as expected. N116C/P248C was significantly more stable than the wild type against chemical and thermal denaturation and more resistant to alpha-chymotrypsin digestion, whereas F163C showed significantly increased thermal stability. All mutants exhibited greater hardness of heat-induced gels than wild type, and in particular, N116C/P248C gave the hardest gel. This result indicates that it is possible to increase hardness of glycinin gel by introduction of cysteine residues using protein engineering.     

Evaluation of the solubility and emulsifying property of soybean proglycinin and rapeseed procruciferin in relation to structure modified by protein engineering

The presence or absence of a highly negatively charged extension region in beta-conglycinin (J. Agric. Food Chem. 1999, 47, 5278) and the length of a highly negatively charged variable region IV in glycinin (J. Agric. Food Chem. 2004, 52, 8197) are important determinants of solubility and emulsifying property. To examine the effects of the variable region IV from proglycinin A1aB1b and A3B4 and of the extension region from beta-conglycinin alpha' (alpha'ext) on solubility and emulsifying properties in detail, several mutants of proglycinin, procruciferin, and beta-conglycinin were designed and prepared in Escherichia coli. Nine out of 10 mutants were expressed at high levels in E. coli and shown to be homotrimer similar to the wild types as assessed by gel filtration. The position of the introduced negatively charged region as well as the amino acid composition were demonstrated to affect solubility at mu = 0.08. All of the proglycinin, procruciferin, and beta-conglycinin mutants with the alpha'ext in the C-terminus, especially the proglycinin mutant, exhibited excellent emulsifying ability and emulsion stability. These indicate that improvement of emulsifying properties by insertion of the alpha'ext in the C-terminus may be generally applicable to seed globulins.     

Grafting of aliphatic and aromatic probes on rapeseed 2S and 12S proteins: influence on their structural and physicochemical properties

Lysyl residues of rapeseed napin (2S) and cruciferin (12S) were acylated and sulfamidated by means of anhydrides and sulfonyl chlorides, respectively. The secondary and tertiary structures as well as the surface hydrophobicity of the modified proteins were studied using circular dichroism, intrinsic fluorescence, and binding of anilinonaphthalenesulfonic acid. The results showed clearly that grafting of hydrophobic chains induced different structural modifications and surface hydrophobicities on the monomeric (2S) and on the hexameric (12S) proteins. Thus, the original structure of the 2S modified protein seemed to be preserved. Therefore, the surface hydrophobicity increased proportionally with the number of groups grafted. Conversely, after modification, 12S was shown to be expanded. As a result, hydrophobic regions were exposed, leading to a much greater hydrophobization of the protein surface. Acylation and sulfamidation appeared, therefore, to be good methods to hydrophobize efficiently the surface of the two proteins and thus might probably induce new functional properties.     

Role of disulfide bonds upon the structural stability of an amaranth globulin.

Analysis of globulin-P, the polymerized amaranth globulin, gave a low amount of free sulfhydryls (10.2 +/- 0.5 micromol/g) from which 7 +/- 1 micromol/g was buried inside the molecule. In addition, its disulfide content was high (51 +/- 1 micromol/g) and similar to soybean 11S globulin content. The more exposed disulfide bridges were found to be stabilizing polymers, whereas the less reactive bridges were either linking P(20) and P(30) polypeptides or forming intrachain linkages. It was found that the buried bonds participate in the stabilization of folded polypeptides and the quaternary structure of the globulin. In turn, the dissociation of polymers and disruption of the quaternary structure by the action of 2-mercaptoethanol reverted upon removal of the reducing agent. This demonstrates that the polymerized state and the quaternary structure of the molecules are most favorable from the thermodynamic point of view. The similar content of SH and SS in globulin-P and globulin-S found in this laboratory suggests that the differences between these proteins may be ascribed to other compositional differences.     

微波对大豆蛋白氧化聚集体结构及功能特性的影响

为了探究不同时间微波处理对大豆蛋白氧化聚集体的结构和功能性质的影响,由偶氮二异丁脒盐酸盐（2,2'-azobis (2-amidinopropane) dihydrochloride,AAPH）诱导构建大豆蛋白氧化反应体系,采用功率为350 W的微波对其照射不同时间(0、10、20、30、40、50、60、70 s),探究微波处理对氧化聚集大豆蛋白的结构特性和加工特性的影响。结果表明,氧化可诱导形成粒径、分子量更大,结构更致密的蛋白质聚集体,同时对加工特性造成损害。适当时间（<30 s）的微波处理会导致氧化聚集体的分子结构打开、粒径降低和浊度降低,无序结构减少,进而改善了起泡性、乳化性和持水、持油性。长处理时间（＞30 s）的微波处理导致已解聚的大豆蛋白分子重新形成更大的分子聚集体,降低功能性质。这表明微波物理场可以通过改变大豆蛋白氧化聚集体的结构和聚集行为调节其功能性质,为大豆蛋白功能性质的改善及微波在大豆蛋白氧化聚集体行为调控的应用方面提供参考。     

大豆丛枝菌根共生结构和多聚磷累积双定位方法

【目的】多聚磷是丛枝菌根内磷的主要贮存形式,定性、定量观察多聚磷对于解析菌根中磷代谢具有重要意义。随着植物体内越来越多的参与菌根真菌与寄主植物之间营养交换过程的基因被鉴定,迫切需要进一步提高根内菌根共生结构和多聚磷累积的染色和定位分析技术。【方法】本研究利用丛枝菌根真菌Glomus mosseae侵染的大豆植株,采集新鲜根样制片,一部分薄根片利用低浓度荧光染料麦胚凝集素,室温染色30 min,在波长488 nm的蓝光激发下使用荧光显微镜观察拍照;另一部分薄根片利用荧光染料4’,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)进行染色,在波长405 nm紫外光激发下观察并拍照;进一步取新鲜制备的薄根片,先后用以上两种荧光染料进行染色,分别在波长405 nm和488 nm的激发光下观察并拍照,完成了菌根共生结构和多聚磷的共定位。【结果】1)使用荧光染料麦胚凝集素,大豆丛枝菌根真菌侵染结构的荧光标记活性染色法,可以清晰地检测到大豆丛枝菌根中所有的共生结构,包括丛枝,泡囊和根内菌丝等。2)在丛枝菌根真菌侵染的根中,各种共生结构都呈现出黄色荧光,为DAPI与多聚磷结合在紫外光激发下的呈色。根段中部分细胞内的蓝白色斑点为DAPI与细胞核中DNA结合的显色结果。在含有成熟丛枝结构的细胞中,也可观察到大部分丛枝呈蓝白色,主要是丛枝膜质结构的呈色。因此,利用荧光染料4’,6-二脒基-2-苯基吲哚二盐酸盐染色法定位多聚磷,能很好地区分多聚磷酸盐、DNA和膜质。3)在以上研究的基础上,通过荧光光路的切换,可以同时观察到菌根共生结构和多聚磷的共定位。处于发育阶段的整个丛枝中多聚磷累积的亮黄色清晰可见。在成熟的丛枝中,由于膜质结构发达,对累积在丛枝结构中的多聚磷的染色观察产生了一定影响,导致仅仅局部的多聚磷累积清晰可见。【结论】本研究建立的大豆菌根共生结构与多聚磷累积的双定位分析系统,能够直观观察植物与丛枝菌根真菌的养分交换,清晰地对丛枝菌根共生结构中多聚磷的累积进行定位分析,可作为从组织和细胞水平研究菌根共生体的重要技术手段。     

Reduction of structural Fe(III) in oxyhydroxides by Shewanella decolorationis S12 and characterization of the surface properties of iron minerals

Purpose  

Dissimilatory iron reduction is an important iron biogeochemical process in subsurface soils. Many researchers have studied the effects of various factors on this process. However, this process in the natural environment is complicated; thus, all the factors should be investigated systematically and simultaneously. The aims of this study were to investigate the effects of Fe(III) availability, surface areas, and crystallinity on Fe(III) reduction in different buffers and to characterize the surface properties of iron minerals.     

植物乳杆菌C88胞外多糖生物合成基因的克隆及序列比对

乳酸菌胞外多糖能显著改善发酵乳制品及食品的流变学和质构特性。为进一步了解乳酸菌胞外多糖的生物合成途径及调控机制,本研究对参与植物乳杆菌C88胞外多糖生物合成基因簇的部分序列进行了克隆和鉴定。根据GenBank中己报道植物乳杆菌基因序列的保守区域设计特异性引物,扩增出植物乳杆菌C88生物合成蛋白基因（cps4A）序列,并通过染色体步移方法克隆了植物乳杆菌C88参与胞外多糖合成基因簇的部分序列（4．9kb）。利用生物信息学方法预测基因簇中6个阅读框的结构和功能,结果表明该序列与己报道的乳酸杆菌胞外多糖生物合成基因具有高度的同源性（〉96％）;对各阅读框功能预测分析发现,这6个基因主要编码参与胞外多糖合成中的多糖合成蛋白、糖链长度检测蛋白、UDP-葡萄糖-4-异构酶和糖基转移酶。本研究将为利用基因工程方法调控多糖的合成和产量提供理论依据。     

Mathematical description of the dynamics of 12C and 14C in the soil humus

Mathematical tools that can be applied to analyze the dynamics of ¹⁴C and ¹²C isotopes in soil humus are discussed. A system of two differential equations is suggested to describe the dynamics of the ¹²C and the ¹⁴C/¹²C ratio (normalized to the NBS standard) with due account for the initial conditions and for the ¹⁴C dynamics in the atmosphere (in particular, the bomb effect). Possible analytical solutions to these equations and algorithms that can be used in the case when these solutions are absent are discussed. Routine problems of the assessment of the dates of soil burying and the coefficient of the humus mineralization on the basis of the experimentally determined ¹⁴C/¹²C ratios in the soil humus are considered with the use of published data. In particular, the specific features of the assessment of the parameters of these equations in the case of the insufficiency of the experimental data are analyzed.     

挤压温度对大豆分离蛋白与原花青素复合物结构和功能特性的影响

为了探究不同挤压温度（40、60、80、100和120℃）对大豆分离蛋白（Soy Isolate Protein,SPI）与葡萄籽原花青素（Grape Seed Proanthocyanidin Extract,GSPE）复合物功能性质及结构特性的影响。该研究以溶解度、乳化性、乳化稳定性、ζ-电位、粒度为指标,利用荧光光谱、红外光谱分析该复合体系中大豆分离蛋白功能性质及结构的变化。结果表明：相较于挤压SPI,经过挤压处理的SPI-GSPE复合物的溶解度、乳化活性指数、乳化稳定性指数、ζ-电位绝对值及持水性均显著提高（P<0.05）,其表面疏水性、持油性显著下降（P<0.05）。随着挤压温度的升高,SPI-GSPE复合物的溶解度、持油性及乳化活性均先增大后减小且在80℃达到最大值,而其表面疏水性先减小后增大且最小值在80℃,ζ-电位绝对值、乳化稳定性及持水性均随温度的升高而降低。粒径分析结果表明,挤压处理后SPI与GSPE形成了更加致密的复合物;荧光光谱及红外光谱结果表明,与GSPE的复合及挤压处理使SPI氨基酸残基所处微环境发生变化,蛋白结构发生变化。以上结果表明挤压温度为80℃时SPI-GSPE复合物功能性质提高幅度最大,为GSPE与SPI复合提高SPI的功能性质提供参考。     

Determination of leghemoglobin components and xylem sap composition by capillary electrophoresis in hypernodulation soybean mutants cultivated in the field

The hypernodulation soybean mutant lines (NOD1-3, NOD2-4, NOD3-7) and their parent Williams, and the mutant En6500 and its parent Enrei were cultivated in a sandy dune field in Niigata, and the nodules and root bleeding xylem sap were sampled at 50, 70, 90, and 120 d after planting (DAP). The nodule size distribution patterns and concentration of leghemoglobin components were determined. The number of nodules of the hypernodulation mutant lines was about two to three times higher than that of the parent lines irrespective of the sampling date. At 50 DAP the nodule size was relatively smaller in the hypernodulation mutant lines, and the total dry weight of the nodules was almost the same in the mutant lines and their parents. At 70 DAP and 90 DAP, the size distribution of the hypernodulation mutant nodules became .almost the same as that of the parent lines, and both the number and total dry weight of the nodules were higher than those of the parent lines. The concentration of four Lb components was separately measured by capillary electrophoresis. The concentration of the Lb components in the hypernodulation mutant lines tended to be lower than in the parents, but the component ratios were not different between the hypernodulation mutants and their parents. Under field conditions, plant growth and nodulation characteristics were more similar between mutants and parents than in the hydroponic culture reported previously, although the mutants did exhibit hypernodulation traits. These findings suggest that the decrease in the Lb concentration and the different Lb components ratios in the mutants may be caused by secondary effects of excess nodulation, such as photosynthate deficiency, rather than by a genetic defect in mutation. The concentration of major nitrogenous compounds (allantoic acid, allantoin, asparagine, aspartic acid, and nitrate) in the xylem sap was also measured by capillary electrophoresis. The concentration of ureides and nitrate in xylem sap decreased with the plant age, but the asparagine concentration increased during the same period. The concentrations of ureides and asparagine were higher, and the nitrate concentration was lower in the mutant lines than in their parents, possibly due to the higher dependence on N₂ fixation than N0₃ ^- utilization. In the xylem sap, nitrate was the major inorganic anion followed by phosphate, sulfate, and chloride, and potassium was the major cation followed by calcium or magnesium and sodium.     

Preparative procedures markedly influence the appearance and structural integrity of protein storage vacuoles in soybean seeds

In legumes, vacuoles serve as the final depository for storage proteins. The protein storage vacuoles (PSVs) of soybean contain electron-transparent globoid regions in which phytic acid ( myo-inositol-1,2,3,4,5,6-hexakisphosphate) is sequestered. This paper reports the effect of preparative procedures on the appearance and ultrastructural integrity of PSVs in soybeans. Electron microscopy examination of both developing and mature soybean seeds that were postfixed with osmium tetroxide revealed PSVs that had a homogeneous appearance with very few globoid crystals dispersed in them. Numerous electron-dense lipid bodies were readily seen in these cells. Omission of osmium tetroxide strikingly altered the appearance of PSVs and aided the visualization of the location of the globoids in the PSVs. In contrast to the osmicated tissue, lipid bodies appeared as electron-transparent spheres. The choice of dehydration reagent or staining procedure had little influence on the appearance of the PSVs. The results of this study demonstrate the profound effect of osmium tetroxide on the appearance and structural integrity of PSVs in soybean.     

Iron deficiency of soybean in the North Central U.S. and associated soil properties

Despite extensive research and variety screening efforts, iron deficiency chlorosis is a common, yield-limiting condition for soybean [Glycine max (L.) Merr.] grown in areas with high pH, calcareous soils. In the North Central U.S., total land area where soybean is grown on high pH soils is approximately 1.8 million ha, with iron deficiency responsible for an estimated loss in soybean grain production of 340,000 Mg at a value of $820 million per annum. This is a significant increase in the extent of iron deficiency problems relative to the past because of an expansion of soybean production in the region. Soil properties associated with iron deficiency in this region compared to adjacent areas without iron deficiency include greater soil moisture content and concentrations of soluble salts, carbonates, and DTPA-Cr, and lesser concentrations of DTPA-Fe, Mn, Ni, and Cd. Iron deficiency occurs due to multiple stresses and not simply to limited available iron. Biotic and management factors such as pests and diseases, symbiotic nitrogen fixation, seeding rate, and herbicide application also interact with iron deficiency in the field. There is a need to better match varieties to the specific soil and environmental conditions to which they are adapted.     

The effect of crop-pasture rotations on the C,N and S contents of soil aggregates and structural stability in a volcanic soil of south-central Chile

Abstract

The effects of six crop-pasture rotations were evaluated on properties of soil aggregates in a volcanic soil (Humic Haploxerand) of south-central Chile. Rotations that included intensive cropping without pastures, and crops with short- or long-term pastures were maintained for 12 years after which soil samples were taken at 0–5 and 5–10 cm depths for analysis of the C, N and S contents in the different grades of water-stable aggregates. The mean weight diameter of the aggregates was also determined as an indicator of structural stability. The results showed that the rotations which included long-term pastures of alfalfa (Medicago sativa L.) or white clover (Trifolium repens L.) had higher contents of C, N and S in soil and there was a higher concentration of these elements in macro-aggregates (>0.5 mm) than in micro-aggregates (<0.5 mm). Additionally, the rotations with pastures also produced greater structural stability of the soil aggregates. Consequently, crop rotations that included pastures, particularly those of longer duration, improved the soil and were therefore a more sustainable use of the soil resource compared to the more intensive rotations. Additionally, there was evidence of hierarchical organization of the soil structure, which is a previously non-described feature of volcanic soils.     

S3307和DTA-6对大豆叶片生理活性及产量的影响

【目的】化控技术是提高作物产量的一项重要技术,研究植物生长调节剂对大豆鼓粒期至成熟期叶片生理活性及产量的影响,旨在探讨调节剂提高大豆产量的作用机理,为调节剂在农业生产上的应用提供科学依据。【方法】于2013 ~ 2014年在黑龙江八一农垦大学林甸试验基地,进行叶面喷施烯效唑(S3307)和2-N, N-二乙氨基乙醇酯(DTA-6)田间试验。试验于初花期(R1期)叶面喷施1次,设3个处理: 1)CK,喷施清水; 2)喷施DTA-6,浓度60 mg/L; 3)喷施S3307,浓度50 mg/L。大豆盛荚期(R4期),用叶绿素仪测定大豆倒3叶和倒9叶的叶绿素值;大豆鼓粒始期(R5期)开始第一次取样,以后每隔5 d 取样一次,共取样7次,测定叶片中的可溶性糖、蔗糖、淀粉和淀粉酶总活性。大豆成熟期测定产量。【结果】在喷植物生长调节剂后22 d,调节剂极显著增加了大豆植株不同部位,尤其是下部叶片的叶绿素含量,S3307和DTA-6处理的倒3叶叶绿素含量比CK增加9.32%、 7.02%,倒9叶叶绿素含量比CK增加11.12%、 10.38%,各处理倒3叶和倒9叶的叶绿素含量与产量及产量构成因素均达到正相关。DTA-6和S3307在喷植物生长调节剂后40、 45和50 d叶片中的可溶性糖含量均高于CK;DTA-6和S3307处理的蔗糖含量分别在喷植物生长调节剂后40~50 d和35~50 d达到了最大增加量,S3307对蔗糖积累的作用效果较好;DTA-6和S3307处理在喷植物生长调节剂后50、55和60 d叶片中的淀粉含量均高于CK;不同调节剂处理对淀粉酶总活性也产生了一定影响,喷植物生长调节剂后第45 d时,调节剂作用的淀粉酶总活性达到最大值,各处理淀粉酶活性的高低顺序为S3307、 DTA-6＞CK均与CK处理差异显著。不同调节剂处理均增加了单株荚数、粒数、粒重,S3307处理的单株荚数和粒数与CK相比,差异均达到了显著水平,DTA-6处理的各产量因素与CK相比,差异均未达到显著水平。2013年S3307和DTA-6处理的产量分别比CK增加20.74%和14.96%;2014年S3307和DTA-6处理的产量分别比CK显著增加19.33%和14.13%。【结论】初花期(R1期)叶面喷施S3307和DTA-6均显著增强了大豆叶片的生理代谢。尤其在喷施调节剂后50 d,此时恰处于籽粒灌浆期,S3307和DTA-6处理的叶片可溶性糖、蔗糖和淀粉含量均高于CK。两调节剂均显著促进了生育后期大豆叶片的生理活性,延缓了叶片衰老,进而提高了大豆产量。     

油酰甘氨酸对C2C12成肌细胞增殖、分化和蛋白质沉积的影响

脂酰氨基酸是一类脂肪酸和氨基酸缩合生成的内源性化合物,在镇痛抗炎、细胞增殖和细胞凋亡等方面具有重要的调控作用.为探讨脂酰氨基酸对骨骼肌发育的影响,本研究以小鼠(Mus musculus)成肌细胞(myoblast cell line,C2C12)为对象,分别采用细胞计数检测法和肌酸激酶活性分析油酰甘氨酸(N-Oleoyl glycine,OLGly)对C2C12细胞的增殖和分化水平的影响.同时用qRT-PCR检测了增殖细胞核抗原、细胞周期素依赖性激酶抑制因子和生肌调节因子5、成肌素的mRNA表达水平.并且,本实验一方面测定了细胞总蛋白含量,另一方面采用Westem blot法检测嘌呤霉素掺入水平,分析了蛋白质沉积相关蛋白以及蛋白质降解相关蛋白的表达水平.结果发现,与无水乙醇对照组相比,基础培养液中分别添加0.2、2和20 μmol/L OLGly对C2C12细胞的数目、肌酸激酶活性以及增殖和分化标志基因表达水平均无显著影响;而蛋白质合成研究结果表明,OLGly可以剂量依赖性提高细胞总蛋白含量(P＜0.05),此外,研究还发现,在OLGly处理组中,细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)蛋白和核糖体蛋白亚型6(ribosomal protein subunit6,rpS6)蛋白的表达水平均呈现剂量和时间依赖性升高(P＜0.05).进一步研究发现,OLGly对蛋白质降解通路相关蛋白的表达无显著性影响.综合上述研究结果可见,OLGly能促进C2C12细胞蛋白质合成,但对其增殖和分化无明显影响.究其机制,可能与ERK和rpS6的蛋白表达水平的上调有关.研究结果为研制调控肌肉发育和蛋白质沉积的新型功能性调控物提供了实验依据.     

Depth distribution of soil organic C and N after long-term soybean cropping in Texas

Crop management practices have potential to enhance subsoil C and N sequestration in the southern U.S., but effects may vary with tillage regime and cropping sequence. The objective of this study was to determine the impacts of tillage and soybean cropping sequence on the depth distribution of soil organic C (SOC), dissolved organic C (DOC), and total N after 20 years of treatment imposition for a silty clay loam soil in central Texas. A continuous soybean monoculture, a wheat–soybean doublecrop, and a sorghum–wheat–soybean rotation were established under both conventional (CT) and no tillage (NT). Soil was sampled after soybean harvest and sectioned into 0–5, 5–15, 15–30, 30–55, 55–80, and 80–105 cm depth intervals. Both tillage and cropping intensity influenced C and N dynamics in surface and subsurface soils. No tillage increased SOC, DOC, and total N compared to CT to a 30 cm depth for continuous soybean, but to 55 cm depths for the more intensive sorghum–wheat–soybean rotation and wheat–soybean doublecrop. Averaged from 0 to 105 cm, NT increased SOC, DOC, and total N by 32, 22, and 34%, respectively, compared to CT. Intensive cropping increased SOC and total N at depths to 55 cm compared to continuous soybean, regardless of tillage regime. Continuous soybean had significantly lower SOC (5.3 g kg⁻¹) than sorghum–wheat–soybean (6.4 g kg⁻¹) and wheat–soybean (6.1 g kg⁻¹), and 19% lower total N than other cropping sequences. Dissolved organic C was also significantly higher for sorghum–wheat–soybean (139 mg C kg⁻¹) than wheat–soybean (92 mg C kg⁻¹) and continuous soybean (100 mg C kg⁻¹). The depth distribution of SOC, DOC, and total N indicated treatment effects below the maximum tillage depth (25 cm), suggesting that roots, or translocation of dissolved organic matter from surface soils, contributed to higher soil organic matter levels under NT than CT in subsurface soils. High-intensity cropping sequences, coupled with NT, resulted in the highest soil organic matter levels, demonstrating potential for C and N sequestration for subsurface soils in the southern U.S.     

13C/12C Ratios of soil organic matter as indicators of vegetation changes in the congo

The ¹³C/¹²C ratios were determined for the organic matter of all horizons of a podzol profile and of the A₁ horizons of some ferrallitic soils, in some grass shoots and in a fossil root fragment from the B_2h horizon of the podzol. The isotope ratio in the organic matter of the A₁ horizon of the podzol matches those in grass shoots from the present savanna vegetation. The ratios in the lower horizons match those of organic matter in the A₁ horizons of soils under forest and that of the fossil root fragment in the B_2h horizon. The ratios thus demonstrate that the humus enrichment of the B_2h horizon of the podzol occurred while it was under forest vegetation and that the present grass vegetation did not take part in the podzolization process. The differences also indicate that savanna replaced forest vegetation after the profile had been formed.