Purification and characterization of assimilatory nitrite reductase from Candida utilis

Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

The regulation of nitrate reductas enad catalase by amino acids in Neurospora crassa

The induction of nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) by nitrate in Neurospora crassa and its control by amino acids have been studied. The growth-inhibitory amino acids, isoleucine and cysteine as well as the growth-promotory ones, glutamine, asparagine, arginine, histidine and NH4+, repress nitrate reductase effectively. Methionine, tryptophan, proline, aspartic acid and glutamic acid exert little control on nitrate reductase. The repression of nitrate reductase by cysteine, isoleucine, glutamine and asparagine is accompanied by inactivation of the enzyme present initially. The nitrate-induced NADPH-cytochrome c reductase (NADPH:cytochrome c oxidoreductase, EC 1.6.2.3) is also repressed by amino acids which control nitrate reductase, providing further evidence to show that these two enzyme activities may reside in the same protein. Catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) has been found to be induced subsequent to the induction of nitrate reductase by nitrate in N. crassa. The induction of catalase is probably by its substrate H2O2 which would be formed by the interaction of the flavine component of nitrate reductase with oxygen. The amino acids which control nitrate reductase, repress catalase also. The catalase level appears to be determined by the nitrate reductase activity of the mycelia.

In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the path way, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.

Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO(2) concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana: a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate. (C) 2010 Elsevier GmbH. All rights reserved.

The NreABC system of Staphylococcus carnosus combines nitrate and oxygen sensing by an NreA,NreB sensor complex

Staphylococcus carnosus is a facultative anaerobic bacterium which features the cytoplasmic NreABC system. It is necessary for regulation of nitrate respiration and the nitrate reductase gene narG in response to oxygen and nitrate availability. NreB is a sensor kinase of a two-component system and represents the oxygen sensor of the system. It binds an oxygen labile [4Fe-4S]2+ cluster under anaerobic conditions. NreB autophosphorylates and phosphoryl transfer activates the response regulator NreC which induces narG expression. The third component of the Nre system is the nitrate receptor NreA. In this study the role of the nitrate receptor protein NreA in nitrate regulation and its functional and physiological effect on oxygen regulation and interaction with the NreBC two-component system were detected. In vivo, a reporter gene assay for measuring expression of the NreABC regulated nitrate reductase gene narG was used for quantitative evaluation of NreA function. Maximal narG expression in wild type S. carnosus required anaerobic conditions and the presence of nitrate. Deletion of nreA allowed expression of narG under aerobic conditions, and under anaerobic conditions nitrate was no longer required for maximal induction. This indicates that NreA is a nitrate regulated inhibitor of narG expression. Purified NreA and variant NreA(Y95A) inhibited the autophosphorylation of anaerobic NreB in part and completely, respectively. Neither NreA nor NreA(Y95A) stimulated dephosphorylation of NreB-phosphate, however. Inhibition of phosphorylation was relieved completely when NreA with bound nitrate (NreA•[NO3-]) was used. The same effects of NreA were monitored with aerobically isolated Fe-S-less NreB, which indicates that NreA does not have an influence on the iron-sulfur cluster of NreB. In summary, the data of this study show that NreA interacts with the oxygen sensor NreB and controls its phosphorylation level in a nitrate dependent manner. This modulation of NreB-function by NreA and nitrate results in nitrate/oxygen co-sensing by an NreA/NreB sensory unit. It transmits the regulatory signal from oxygen and nitrate in a joint signal to target promoters. Therefore, nitrate and oxygen regulation of nitrate dissimilation follows a new mode of regulation not present in other facultative anaerobic bacteria.

Sulfate and Nitrate Assimilation in Leaves of Quercus ilex and Quercus pubescens Grown Near Natural CO2 Springs in Central Italy

The effect of long-term exposure to elevated pCO2 concentrations on sulfate and nitrate assimilation was studied under field conditions using leaves from Quercus ilex and Quercus pubescens trees growing with ambient or elevated CO2 concentrations in the vicinity of three natural CO2 springs, Bossoleto, Laiatico and Sulfatara, in Tuscany, Italy. The activity of the key enzymes of sulfate assimilation, adenosine 5′-phosphosulfate reductase (APR) and nitrate assimilation, nitrate reductase (NR), were measured together with the levels of acid soluble thiols, and soluble non-proteinogenic nitrogen compounds. Whereas NR activity remained unaffected in Q. ilex or increased Q. pubescence, APR activity decreased in the area of CO2 springs. The latter changes were often accompanied by increased GSH concentrations, apparently synthesized from H2S and SO2 present in the gas mixture emitted from the CO2 springs. Thus, the diminished APR activity in leaves of Q. ilex and Q. pubescence from spring areas can best be explained by the exposure to gaseous sulfur compounds. Although the concentrations of H2S and SO2 in the gas mixture emitted from the vents at the CO2 springs were low at the Bossoleto and Laiatico spring, these sulfur gases pose physiological effects, which may override consequences of elevated pCO2.

Dual pathways for regulation of root branching by nitrate

Root development is extremely sensitive to variations in nutrient supply, but the mechanisms are poorly understood. We have investigated the processes by which nitrate (NO3−), depending on its availability and distribution, can have both positive and negative effects on the development and growth of lateral roots. When Arabidopsis roots were exposed to a locally concentrated supply of NO3− there was no increase in lateral root numbers within the NO3−-rich zone, but there was a localized 2-fold increase in the mean rate of lateral root elongation, which was attributable to a corresponding increase in the rate of cell production in the lateral root meristem. Localized applications of other N sources did not stimulate lateral root elongation, consistent with previous evidence that the NO3− ion is acting as a signal rather than a nutrient. The axr4 auxin-resistant mutant was insensitive to the stimulatory effect of NO3−, suggesting an overlap between the NO3− and auxin response pathways. High rates of NO3− supply to the roots had a systemic inhibitory effect on lateral root development that acted specifically at the stage when the laterals had just emerged from the primary root, apparently delaying final activation of the lateral root meristem. A nitrate reductase-deficient mutant showed increased sensitivity to this systemic inhibitory effect, suggesting that tissue NO3− levels may play a role in generating the inhibitory signal. We present a model in which root branching is modulated by opposing signals from the plant’s internal N status and the external supply of NO3−.

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature1

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m−2 s−1) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m−2 s−1) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.

Wheat leaves emit nitrous oxide during nitrate assimilation

Nitrous oxide (N2O) is a key atmospheric greenhouse gas that contributes to global climatic change through radiative warming and depletion of stratospheric ozone. In this report, N2O flux was monitored simultaneously with photosynthetic CO2 and O2 exchanges from intact canopies of 12 wheat seedlings. The rates of N2O-N emitted ranged from <2 pmol⋅m−2⋅s−1 when NH\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{4}^{+}}}\end{equation*}\end{document} was the N source, to 25.6 ± 1.7 pmol⋅m−2⋅s−1 (mean ± SE, n = 13) when the N source was shifted to NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}. Such fluxes are among the smallest reported for any trace gas emitted by a higher plant. Leaf N2O emissions were correlated with leaf nitrate assimilation activity, as measured by using the assimilation quotient, the ratio of CO2 assimilated to O2 evolved. 15N isotopic signatures on N2O emitted from leaves supported direct N2O production by plant NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} assimilation and not N2O produced by microorganisms on root surfaces and emitted in the transpiration stream. In vitro production of N2O by both intact chloroplasts and nitrite reductase, but not by nitrate reductase, indicated that N2O produced by leaves occurred during photoassimilation of NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} in the chloroplast. Given the large quantities of NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} assimilated by plants in the terrestrial biosphere, these observations suggest that formation of N2O during NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} photoassimilation could be an important global biogenic N2O source.

Effects of nitrogen sources on the nitrate assimilation in Haloferax mediterranei: growth kinetics and transcriptomic analysis

The haloarchaeon Haloferax mediterranei is able to grow in a defined culture media not only in the presence of inorganic nitrogen salt but also with amino acid as the sole nitrogen source. Assimilatory nitrate and nitrite reductases, respectively, catalyze the first and second reactions. The genes involved in this process are nasA, which encodes nitrate reductase and is found within the operon nasABC, and nasD, which encodes nitrite reductase. These genes are subjected to transcriptional regulation, being repressed in the presence of ammonium and induced with either nitrate or nitrite. This type of regulation has also been described when the amino acids are used as nitrogen source in the minimal media. Furthermore, it has been observed that the microorganism growth depends on nitrogen source, obtaining the lowest growth rate in the presence of nitrate and aspartate. In this paper, we present the results of a comparative study of microorganism growth and transcriptomic analysis of the operon nasABC and gene nasD in different nitrogen sources. The results are the first ever produced in relation to amino acids as nitrogen sources within the Halobacteriaceae family.

Dimethylsulfide dehydrogenase from rhodovulum sulfidophilum: EPR spectroscopy and biochemical analysis reveal its place in the DMSO reductase family of molybdenum enzymes

Dimethylsulfide (DMS) dehydrogenase catalyses the oxidation of DMS to dimethylsulfoxide. The purified enzyme has three subunits of Mr = 94, 38 and 32 kDa and has an optical spectrum dominated by a b-type cytochrome. The metal ion and nucleotide analysis revealed 0.5 g-atom Mo, 9.8 g-atom Fe and 1.96 mol GMP per tool of enzyme. Taken together, these data indicate that DMS dehydrogenase contains a bis(MGD)Mo cofactor. A comparison of the Nterminal amino acid sequence of DMS dehydrogenase revealed that the Mo-containing ct-subunit was most closely related to the c~-subunits of nitrate reductase (NarG) and selenate reductase (SerA). Similarly, the [~-subunit of DMS dehydrogenase was most closely related to the [3-subunits of nitrate reductase (NarH) and selenate reductase (SerB). Variable temperature X-band EPR spectra (120-2K) of 'as isolated' DMS dehydrogenase showed resonances arising from multiple redox centres, Mo(V), [3Fe-4S] +, [4Fe-4S] ÷. A pH dependent EPR study of the Mo(V) centre in lH20 and 2H20 reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(V)-X and Mo(V)-OH. Between pH6 and 8.2 the dominant species is Mo(V)-OH2 and Mo(V)-X is a minor component. X is probably the anion, chloride. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other Mo(V) centres in metalloproteins showed that it was most similar to the low pH nitrite spectrum of E. coli nitrate reductase (NarGHI). The spin Hamiltonian parameters (2.0158, 1.8870, 1.8620) for the [4Fe-4S] + cluster suggests the presence of histidine (N) coordination to iron in this cluster. It is suggested that this unusual [Fe-S] cluster may be associated with a histidine-cysteine rich sequence at the N-terminus of the ct-subunit of DMS dehydrogenase.

The effect of nitrate and phosphate availability on Emiliania huxleyi(NZEH) physiology under different CO2 scenarios

Growth and calcification of the marine coccolithophorid Emiliania huxleyi is affected by ocean acidification and macronutrients limitation and its response varies between strains. Here we investigated the physiological performance of a highly calcified E. huxleyi strain, NZEH, in a multiparametric experiment. Cells were exposed to different CO2 levels (ranging from 250 to 1314 µatm) under three nutrient conditions [nutrient replete (R), nitrate limited (-N), and phosphate limited (-P)]. We focused on calcite and organic carbon quotas and on nitrate and phosphate utilization by analyzing the activity of nitrate reductase (NRase) and alkaline phosphatase (APase), respectively. Particulate inorganic (PIC) and organic (POC) carbon quotas increased with increasing CO2 under R conditions but a different pattern was observed under nutrient limitation. The PIC:POC ratio decreased with increasing CO2 in nutrient limited cultures. Coccolith length increased with CO2 under all nutrient conditions but the coccosphere volume varied depending on the nutrient treatment. Maximum APase activity was found at 561 ?atm of CO2 (pH 7.92) in -P cultures and in R conditions, NRase activity increased linearly with CO2. These results suggest that E. huxleyi's competitive ability for nutrient uptake might be altered in future high-CO2 oceans. The combined dataset will be useful in model parameterizations of the carbon cycle and ocean acidification.

一氧化氮在磷酸盐调节拟南芥根系形态变化中的作用

磷缺乏已成为制约世界农业生产的重要因子。植物根系的大小和形态是决定植物吸收土壤磷能力的重要因素，而且根系的生长发育与磷素的分布及其有效性密切相关。关于磷酸盐调节植物根系生长研究已有很多报道，但其生理和分子机制仍不清楚。一氧化氮 (NO) 是一种重要的气体信号分子，参与调控植物的生长发育和对多种逆境胁迫的应答反应。本文选用拟南芥为实验材料，研究探讨了NO与缺磷诱导的拟南芥根系形态变化之间的关系，主要结果如下： 用正常磷水平 (1 mM) 和低磷水平 (1 µM) 处理拟南芥幼苗，发现低磷抑制主根伸长，刺激侧根发生。外源NO供体销普纳 (SNP) 也抑制主根、刺激侧根生长，与低磷诱导根系形态变化相似。NO清除剂c-PTIO和一氧化氮合成酶 （NOS）抑制剂L-NNA均可部分减缓由低磷引起的对主根生长的抑制和对侧根的刺激作用。暗示低磷诱导的拟南芥根系形态的变化可能与NO含量的降低有关。 利用NO荧光标记物DAF-FM和激光共聚焦显微成像技术，本研究发现缺磷6 h和24 h后根细胞内源NO含量显著增加，而且NOS 抑制剂能减少低磷诱导的根细胞NO含量的增加。与正常供磷处理相比，低磷处理6 h和24 h，拟南芥根中编码与NO合成相关的基因（AtNOA1）的表达量增加，缺磷24 h后根中NOS酶活性升高。为了明确低磷诱导的NO 增加是否与硝酸还原酶（NR）介导的NO合成有关，本论文进一步研究了低磷对拟南芥硝酸还原酶活性和编码NR基因 （AtNR1和AtNR2）表达的影响。研究发现低磷处理6 h和24 h后和AtNR1和AtNR2基因的表达均没有变化，且蛭石中生长的拟南芥缺磷1个月后NR活性也没有发生变化;拟南芥的NR双突变体nia1，nia2在低磷处理24 h后，其根中的内源NO含量表现出与野生型相同的增加。因此这些研究结果表明，缺磷后拟南芥根细胞NO的含量增加主要由于NOS的活性升高，而与NR介导的NO合成无关。 已有资料表明低磷诱导植物根细胞内源过氧化氢（H2O2）分布和含量的变化。本论文研究了低磷处理对用H2O2标记物CM-H2DCFDA标记不同磷处理下的拟南芥根中的H2O2。研究发现，缺磷6 h根中H2O2的分布无明显变化，缺磷24 h后H2O2呈斑块状分布，且多集中在根尖伸长区。缺磷24 h后，叶片中的抗氧化保护酶—超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性没有明显变化。说明缺磷24 h 后产生的H2O2没有引起氧化胁迫，而是作为一种信号分子，与NO相互作用共同介导低磷胁迫的应答反应。关于NO与H2O2在低磷诱导的根形态变化中的信号转导过程还有待进一步研究。

增强UV-B辐射下一氧化氮对小球藻氮素代谢和类囊体的保护作用

由于到达地球表面的紫外线B辐射不断加强,生物生长受到了威胁。UV-B的增强改变了生物体赖以生存的环境,影响了藻类生物生长,抑制了其光合作用。以BG11为培养基,在室内培养的条件是光照强度为60μmol·m-2s-(1昼夜比为12h∶12h),温度为26℃,研究了一氧化氮(NO)在增强UV-B(强度为0.2J·m-2s-1)辐射下的对小球藻的作用。测定了小球藻的硝酸还原酶(NR,nitrate reductase)、亚硝酸还原酶(NiR,nitrite reductase)、谷胱甘肽还原酶(GS,gluta

EFFECTS OF IRON ON PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISTICS OF Microcystis wesenbergii (Kom.) Kom. (Cyanobacterium)

Iron is an essential trace element for biological requirements of phytoplankton. Effects of iron on physiological and biochemical characteristics of Microcystis wesenbergii were conducted in this study. Results showed that 0.01 mu M [Fe3+] seriously inhibited growth and chlorophyll synthesis of M. wesenbergii, and induced temporary increase of ATPase activities, however, NR. ACP and ALP activities were restrained by iron limitation. Interestingly, iron addition on day 8 resulted in the gradual restoration of structures and functions of above enzymes and resisted a variety of stresses from iron limitation. M. wesenbergii in 10 mu M [Fe3+] treatment group grew normally. enzymes maintained normal levels, and residual phosphate contents in cultures first sharply decreased, then smoothly as M. wesenbergii has a characteristic of luxury consumption of phosphorus. Above parameters in 100 mu M [Fe3+] treatment group were almost same with those in 10 mu M [Fe3+] treatment group except for NR, ACP and ALP activities. In 100 mu M [Fe3+] treatment group, activities of ACP and ALP had temporary increase because phosphate and ferric iron could form insoluble compound - ferric phosphate (Fe3PO4) through adsorption effect. resulting in lack of bioavailable phosphate in culture media. The experiment suggested that too low or too high iron can affect obviously physiological and biochemical characteristics of M. wesenbergii.