Photosynthetic rate,chlorophyll fluorescence parameters,and lipid peroxidation of maize leaves as affected by zinc deficiency

Pot trial in greenhouse was conducted using cumulic cinnamon soil from North China to study the effects of zinc deficiency on CO₂ exchange, chlorophyll fluorescence, the intensity of lipid peroxidation, and the activity of superoxide dismutase (SOD) in leaves of maize seedlings. Zn deficiency resulted in a reduction of net photosynthetic rate and stomatal conductance to H₂O. The maximum quantum efficiency of photosystem 2 (PS2) and the PS2 activity were depressed, while the pool size of the plastoquinone molecules was not affected by Zn deficiency. The content of super oxygen anion radical (O₂ ^·−) and the intensity of lipid peroxidation as assessed by malonyldialdehyde content in Zn-deficient leaves were higher than those in Zn-sufficient leaves. The activity of SOD increased with Zn application. The adverse influence of Zn-deficiency on the light stage of photosynthesis is probably one of possible reasons for the limitation of photosynthetic capacity in maize leaves.     

Zinc uptake by rice,as affected by metabolic inhibitors and competing cations

Summary The effects of zinc application on zinc uptake, distribution and translocation in maize and barley grown in zinc deficient soil with high pH and high calcium content were studied. Zn⁶⁵ content and uptake in roots, sheaths and blades of maize and barley plants increased significantly with increased levels of zinc application. The sheaths contained highest Zn⁶⁵ content followed by roots and blades. The distribution of total zinc, however, differed from that of radioactive zinc. The roots had the highest zinc content, followed by sheaths and blades. The two species differed very little in zinc distribution patterns. The autoradiographs of intact maize and barley plants showed that Zn⁶⁵ was fairly evenly distributed in the main and auxiliary roots, but, there was a relatively higher concentration at the root-stem junction. The Zn⁶⁵ concentration was higher in nodes than in internodes, and in young emerging leaves compared to older leaves. re]19730819 Institute of Soil Science and Isotope Laboratory Agricultural University of Norway     

外源NO对缺镁胁迫下玉米幼苗生长和离子平衡的影响

研究了在缺镁胁迫下,外源NO对缺镁玉米幼苗生长、根系活力和离子含量的影响。结果表明,缺镁胁迫使玉米幼苗株高、根长和干鲜重下降,根系活力降低,N元素在地上部和根部分配失调,新叶和老叶中Mg2＋、Cu2＋、Fe3＋、Mn2＋等离子含量下降,Ca2＋、K＋、Zn2＋等离子含量上升。根中Mg2＋离子含量下降,Ca2＋、K＋、Zn2＋、Cu2＋、Fe3＋、Mn2＋等离子含量上升。用100μmol·L-1一氧化氮供体硝普钠（SNP）处理后,玉米幼苗株高、根长、干重和鲜重均提高,根系活力增强,改善了N代谢,新叶中Ca2＋、K＋和Zn2＋等离子含量下降,Mg2＋、Cu2＋、Fe3＋和Mn2＋等离子含量提高,老叶中Mg2＋、Ca2＋、K＋和Zn2＋等离子含量下降,Cu2＋、Fe3＋和Mn2＋等离子含量提高,根中Mg2＋、Ca2＋、K＋、Cu2＋、Zn2＋、Fe3＋和Mn2＋离子含量均下降。实验结果表明,NO保护玉米幼苗免受缺镁胁迫的影响。     

Probing the role of tryptophan-derived secondary metabolism in defense responses against Bipolaris oryzae infection in rice leaves by a suicide substrate of tryptophan decarboxylase

Tryptophan-derived secondary metabolites, including serotonin and its hydroxycinnamic acid amides, markedly accumulate in rice leaves in response to pathogen attack. These compounds have been implicated in the physical defense system against pathogen invasion by being deposited in cell walls. Serotonin is biosynthesized from tryptophan via tryptamine, and tryptophan decarboxylase (TDC) catalyzes the first committed reaction. In this study, (S)-α-(fluoromethyl)tryptophan (S-αFMT) was utilized to investigate the effects of the inhibition of TDC on the defense responses of rice leaves. S-αFMT, enantiospecifically synthesized from l-tryptophan, effectively inhibited TDC activity extracted from rice leaves infected by Bipolaris oryzae. The inhibition rate increased dependently on the incubation time, indicating that S-αFMT served as a suicide substrate. Treatment of rice seedlings with S-αFMT suppressed accumulation of serotonin, tryptamine, and hydroxycinnamic acid amides of serotonin in a dose-dependent manner in B. oryzae-inoculated leaves. The lesions formed on seedlings treated with S-αFMT lacked deposition of brown materials, and those leaves were severely damaged in comparison with leaves without S-αFMT treatment. Administrating tryptamine to S-αFMT-treated leaves restored accumulation of tryptophan-derived secondary metabolites as well as deposition of brown material. In addition, tryptamine administration reduced damage caused by fungal infection. Accordingly, the accumulation of tryptophan-derived secondary metabolites was suggested to be part of the effective defense mechanism of rice.     

Effect of a Photo-synthetic Inhibitor on Tryptamine Pathway-mediated Sekiguchi Lesion Formation in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

A lesion-mimic mutant of rice (cv. Sekiguchi-asahi) showed enhanced resistance to Magnaporthe grisea infection, thereby inducing Sekiguchi lesion ( sl ) formation and tryptamine accumulation under light. Both Sekiguchi lesion formation and tryptamine accumulation in leaves infected with M. grisea were inhibited by pretreatment with the photosynthetic inhibitor, 3-(3, 4-Dichlorophenyl)-1,1-dimethylurea (DCMU), which suppressed the gene expression of tryptophan decarboxylase ( TDC ), monoamine oxidase activity, H₂O₂ generation and DNA fragmentation. Catalase activity was inhibited by M. grisea infection under light, but magnitude of the inhibition was reduced in leaves pretreated with DCMU. Furthermore, tryptophan accumulated in M. grisea- infected leaves under light but not in DCMU-pretreated ones. Interestingly, such DCMU inhibition was reduced in the presence of tryptophan. Our studies suggest that chloroplasts function as the inhibitor of anti-oxidant system such as catalase activity and the supplier of a precursor of tryptamine and tryptophan in the sl mutant infected with M. grisea .     

AN ASSAY PROCEDURE FOR TRYPTAMINE IN BRAIN AND SPINAL CORD USING ITS [3H]DANSYL DERIVATIVE

—The tryptamine content of rat and mouse brain and spinal cord was determined with a radiochemical derivative assay, using [³H]dansyl chloride. The amine was extracted into toluene-isoamyl alcohol, back-extracted into dilute acid, then adsorbed onto a non-ionic polystyrene resin, and dansylated in tetrahydrofuran after elution from the resin. Optimum recoveries were obtained with TCA extracts, although significant losses occurred due to surface adsorption and protein binding. The brain content of tryptamine increased after MAO inhibition and was not significantly further increased when tryptophan loading was combined with inhibition of MAO and/or tryptophan 5-hydroxylase. The tryptamine concentration of spinal cord exceeded that of brain and increased rapidly after death. Among brain regions tryptamine concentrations were greatest in hypothalamus and striatum and lowest in cerebral cortex and cerebellum.     

High levels of tryptamine accumulation in transgenic tobacco expressing tryptophan decarboxylase

A full-length complementary DNA clone encoding tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus (De Luca V, Marineau C, Brisson N [1989] Proc Natl Acad Sci USA 86: 2582-2586) driven by the CaMV 35S promoter was introduced into tobacco (Nicotiana tabacum) to direct the synthesis of the protoalkaloid tryptamine from endogenous tryptophan. Young, fully expanded leaves of CaMV 35S-TDC transformed plants had from four to 45 times greater TDC activity than did controls. Tryptamine accumulated in transgenic plants to levels that were directly proportional to their TDC specific activity. Despite their increased tryptamine content, the growth and development of the CaMV 35S-TDC plants appeared normal with no significant differences in indole-3-acetic acid levels between high tryptamine and control plants. Plants with the highest TDC activity contained more than 1 milligram of tryptamine per gram fresh weight, a 260-fold increase over controls.     

Ribonuclease activity as an index of hidden hunger of zinc in crops

Summary The activity of RN-ase at zero zinc level was found to be 2, 173-1 and 2 times higher in all the varieties of rice (Jaya and HM484) and Maize (Ganga 5 and Vijay) than that of 5 ppm zinc at 15/20 days, 30/40 days and 45/60 days growth stages respectively, but the difference in the zinc content of leaves between two treatments was not found to be significant except that of 30/40 days growth stage. Similarly the potash content in both the treatments did not vary, but the dry matter production of whole plant and protein content of leaves except 15/20 days growth stage varied significantly. The data suggest thirty ppm P released per 100 g protein per hour (RN-ase activity) could be taken as threshold value above which hidden deficiency at an early growth stage is detected. Also an average concentration of 27 and 25 ppm zinc in rice and maize leaves respectively could be taken as critical limit for zinc deficiency at their 15/20 days of growth respectively. On the basis of RN-ase activity the hidden hunger of zinc in crop can be predicted at an early growth stage (15/20 days) where the zinc content in plant tissue fail to reveal the true picture of zinc deficiency.     

Accumulation of Indole‐3‐Acetic Acid in Rice sl Mutant Leaves Infected with Bipolaris oryzae

Rice leaves accumulate serotonin in response to infection by Bipolaris oryzae. The leaves of the sl mutant, which is deficient in the gene encoding tryptamine 5‐hydroxylase, accumulate tryptamine instead of serotonin upon infection by B. oryzae. Because tryptamine is a possible precursor of indole‐3‐acetic acid (IAA), we investigated the accumulation of IAA in sl leaves infected with B. oryzae. Liquid chromatography coupled with tandem mass spectrometry analysis indicated that IAA accumulated at approximately 1.5 μmol/gFW in the leaves of sl mutant. This accumulation was suppressed by 95% by the treatment with the tryptamine decarboxylase inhibitor, (S)‐α‐(fluoromethyl)tryptophan, at 100 μm , indicating that tryptamine served as the precursor of IAA. The accumulation of IAA was not reproduced by treatment with CuCl₂ or by exogenous feeding of tryptamine. Furthermore, inoculation of Magnaporthe grisea induced only a lower level of IAA accumulation. On the other hand, B. oryzae produced IAA in culture media containing tryptamine. These findings strongly suggested that the metabolism of tryptamine by B. oryzae was responsible for IAA accumulation in the leaves of the sl mutant. Serotonin added to the culture media was also converted into 5‐hydroxyindole‐3‐acetic acid (5HIAA) at a rate similar to that of tryptamine. Considering that wild‐type rice leaves accumulate serotonin for defensive purposes, reducing the concentration of serotonin by conversion into 5HIAA may be significant as a detoxification process in the interaction between B. oryzae and rice.     

Estimation of physiologically active zinc in maize by biochemical assay

Summary The enzymes ribonuclease, aldolase and carbonic anhydrase were evaluated as biochemical assays for physiologically active zinc in maize. Seedlings were cultured for 14 or 30 days on a black-earth soil with factorial combinations of phosphorus and zinc fertilizers so as to produce in the leaves varying levels of active zinc at a constant level of total zinc. Enzyme activity was correlated with plant growth, leaf nutrient composition and the occurrence of visual symptoms of zinc deficiency. Ribonuclease and aldolase activities in leaf tissues were insensitive to the changes in active zinc concentration, though aldolase activity was sensitive to phosphorus deficiency. In contrast, leaf carbonic anhydrase activity correlated well with the onset and correction of zinc deficiency symptoms and, as early as 14 days after emergence, was more sensitive to the deficiency than was plant growth. The sensitivity of carbonic anhydrase to changes in active zinc and its specificity for such changes were further examined in 10 to 30 day-old plants grown in solution culture. Zinc was added to plants which had been cultured without zinc for 16 days. A 2.5-fold increase in carbonic anhydrase activity preceded responses by either zinc concentration or plant growth. We propose the use of leaf carbonic anhydrase as an index of active zinc in maize, particularly to supplement inorganic analysis in the diagnosis of zinc deficiency when much of the zinc in the plants is inactive. The assay is simple, is sensitive to and specific for zinc status, and enables early detection of a deficiency before irreversible biochemical events predispose a large yield reduction.     

Induction of N-hydroxycinnamoyltyramine synthesis and tyramine N-hydroxycinnamoyltransferase (THT) activity by wounding in maize leaves

Both N-p-coumaroyl- and N-feruloyltyramine accumulated in response to wounding in leaf segments of maize. The amount of N-hydroxycinnamoyltyramines started to increase 3-6 h after wounding and peaked at 12 h. Thereafter, the amount of N-p-coumaroyltyramine decreased rapidly, while the N-feruloyltyramine content remained at a high level. The accumulation of N-hydroxycinnamoyltyramines was accompanied by an increase in the tyramine N-hydroxycinnamoyltransferase (THT) activity. This increase was initially detected 3 h after wounding and reached a maximum at 36 h, the level of activity being 40 and 11 times that in the leaves before wounding and in the control leaves, respectively. Partial purification of THT from wounded leaves by (NH4)2SO4 precipitation and subsequent two steps of anion-exchange chromatography resulted in a 12.5-fold increase in specific activity. Kinetic studies with this partially purified enzyme revealed that the best substrates were tyramine and feruloyl-CoA, although tryptamine and sinapoyl-CoA also efficiently served as substrates. The apparent native molecular weight of the enzyme was determined by gel filtration as 40 kDa.     

玉米、小麦与花生间作改善花生铁营养机制的探讨

采用土培盆栽方法模拟研究了玉米／花生、小麦／花生间作对花生铁营养状况的影响及其作用机制。结果表明,禾本科作物与花生间作对花生的铁营养状况有显著影响：当花生与玉米或小麦分别间作时,花生新叶叶色正常,而花生单作则表现出严重的缺铁黄化现象,间作花生新叶活性铁、叶绿素含量明显高于单作,两种间作花生各部位铁含量和吸收量明显高于单作,间作明显地促进了铁向花生地上部的转移;在单作花生表现缺铁症状１４ｄ的时间范围内,其根系质外体铁含量仅是间作花生的５２％～８０％;而根系还原力则是单作花生在表现缺铁症状后迅速提高,至缺铁第６ｄ时还原力达到最大值,随后花生根系还原力迅速下降,而间作花生在０～１４ｄ内还原力增加速度缓慢,在１０～１４ｄ中其根系还原力明显地高于单作花生根系还原力。其主要原因可能是禾本科作物玉米、小麦根系分泌物（如：麦根酸类植物铁载体）螯合土壤中难溶性铁并被花生吸收利用。     

Temporal profiling of essential amino acids in developing maize kernel of normal,opaque - 2 and QPM germplasm

Maize, an important cereal crop, has a poor quality of endosperm protein due to the deficiency of essential amino acids, especially lysine and tryptophan. Discovery of mutants such as opaque-2 led to the development of nutritionally improved maize with a higher concentration of lysine and tryptophan. However, the pleiotropic effects associated with opaque-2 mutants necessitated the development of nutritionally improved hard kernel genotype, the present-day quality protein maize (QPM). The aim of present study was to analyze and compare the temporal profile of lysine and tryptophan in the developing maize kernel of normal, opaque-2 and QPM lines. A declining trend in protein along with tryptophan and lysine content was observed with increasing kernel maturity in the experimental genotypes. However, opaque-2 retained the maximum concentration of lysine (3.43) and tryptophan (1.09) at maturity as compared to QPM (lysine-3.05, tryptophan-0.99) and normal (lysine-1.99, tryptophan-0.45) lines. Opaque-2 mutation affects protein quality but has no effect on protein quantity. All maize types are nutritionally rich at early stages of kernel development indicating that early harvest for cattle feed would ensure a higher intake of lysine and tryptophan. Two promising lines (CML44 and HKI 1105) can be used for breeding high value corn for cattle feed or human food in order to fill the protein inadequacy gap. Variation in lysine and tryptophan content within QPM lines revealed that differential expression of endosperm modifiers with varying genetic background significantly affects nutritional quality, indicating that identification of alleles affecting amino acid composition can further facilitate QPM breeding program.     

Increased tryptophan decarboxylase and monoamine oxidase activities induce Sekiguchi lesion formation in rice infected with Magnaporthe grisea

Sekiguchi lesion (sl)-mutant rice infected with Magnaporthe grisea showed increased light-dependent tryptophan decarboxylase (TDC) and monoamine oxidase (MAO) activities. TDC and MAO activities were observed before the penetration of M. grisea to rice cells and maintained high levels even after Sekiguchi lesion formation. Light-dependent expression of TDC gene was observed in leaves inoculated with M. grisea before Sekiguchi lesion formation. Spore germination fluid (SGF) of M. grisea also induced Sekiguchi lesion formation accompanied by increased enzymes activities and tryptamine accumulation. Sekiguchi lesion was also induced by treatments with tryptamine and beta-phenylethylamine, which are substrates for MAO, but was not induced by non-substrates such as indole-3-propionic acid, (+/-)-phenylethylamine and tryptophan under light. Light-dependent induction of Sekiguchi lesion by tryptamine was significantly inhibited in the presence of MAO inhibitors, metalaxyl and semicarbazide, and H2O2-scavengers, ascorbic acid and catalase. H2O2 in M. grisea-infected leaves with and without Sekiguchi lesions was demonstrated directly in situ by strong 3,3'-diaminobenzidine (DAB) staining. On the other hand, H2O2 induced Sekiguchi lesions on leaves of cv. Sekiguchi-asahi under light, but not in darkness. This difference was associated with the decrease of catalase activity in infected leaves under light and the absence of decrease in darkness. We hypothesize that the H2O2-induced breakdown of cellular organelles such as chloroplasts and mitochondria in mesophyll cells may cause high TDC and MAO activities and the development of Sekiguchi lesion, and that the sl gene products in wild-type rice may function as a suppressor of organelle breakdown caused by chemical or environmental stress.     

The indole alkaloid tryptamine produced in transgenic Petunia hybrida

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine, an anti-insect compound. This TDC cDNA was transformed and expressed in transgenic Petunia hybrida under the control of the strong and constitutive 35S promoter from cauliflower mosaic virus. Kanamycin screening and Southern hybridization with the TDC cDNA confirmed plant transformation. Northern analysis indicated greater TDC mRNA accumulation in transgenic plants compared to non-transformed plants. Additionally, eight-fold more tryptamine accumulated in leaves of kanamycin resistant transgenic plants compared to non-transformed plants. Flower petals from the transgenic plants contained lower tryptamine levels than their leaves. Because tryptamine titers were higher in transformed plants compared to controls, over-expression of the TDC enzyme may partially overcome endogenous tryptamine catabolism and/or other negative biosynthetic regulation. Future alteration of tryptamine breakdown in Petunia may further increase total endogenous tryptamine concentrations, potentially discouraging insect reproduction on these transgenic plants.     

Dependence of photosynthesis of sunflower and maize leaves on phosphate supply, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and ribulose-1,5-bisphosphate pool size

Sunflower (Helianthus annuus L. cv Asmer) and maize (Zea mays L. cv Eta) plants were grown under controlled environmental conditions with a nutrient solution containing 0, 0.5, or 10 millimolar inorganic phosphate. Phosphate-deficient leaves had lower photosynthetic rates at ambient and saturating CO₂ and much smaller carboxylation efficiencies than those of plants grown with ample phosphate. In addition, phosphate-deficient leaves contained smaller quantities of total soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit area, although the relative proportions of these components remained unchanged. The specific activity of Rubisco (estimated in the crude extracts of leaves) was significantly reduced by phosphate deficiency in sunflower but not in maize. Thus, there was a strong dependence of carboxylation efficiency and CO₂-saturated photosynthetic rate on Rubisco activity only in sunflower. Phosphate deficiency decreased the 3-phosphoglycerate and ribulose-1,5-bisphosphate (RuBP) contents of the leaf in both species. The ratio of 3-phosphoglycerate to RuBP decreased in sunflower but increased in maize with phosphate deficiency. The calculated concentrations of RuBP and RuBP-binding sites in the chloroplast stroma decreased markedly with phosphate deficiency. The ratio of the stromal concentration of RuBP to that of RuBP-binding sites decreased in sunflower but was not affected in maize with phosphate deficiency. We suggest that a decrease in this ratio made the RuBP-binding sites more vulnerable to blockage or inactivation by tight-binding metabolites/inhibitors, causing a decrease in the initial specific activity of Rubisco in the crude extract from phosphate-deficient sunflower leaves. However, the decrease in Rubisco specific activity was much less than the decrease in the RuBP content in the leaf and its concentration in the stroma. A large ratio of RuBP to RuBP-binding sites may have maintained the Rubisco-specific activity in phosphate-deficient maize leaves. We conclude that the effect of phosphate deficiency is more on RuBP regeneration than on Rubisco activity in both sunflower and maize.     

Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxlyase results in a dark brown phenotype and stunted growth

A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxlyase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxlyase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5′ hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, dark-brown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the dark-brown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.     

锌对不同基因型玉米幼苗碳氮代谢的影响

选用不同耐锌玉米品种‘四单19’（低锌敏感型）和‘牡丹9’（低锌不敏感型）,采用营养液水培,研究不同锌浓度对碳酸酐酶（CA）活性、光合特性、可溶性糖含量等的影响。结果表明：玉米对锌具有选择运输的能力,当锌浓度过大对幼苗造成伤害时,根系吸收的锌向地上部运输的比例减少,以减少过量的锌对地上部造成的伤害。锌与CA活性密切相关,而CA活性与净光合速率的变化并非密切相关。低锌和高锌均显著增加了‘四单19’的可溶性糖含量,而‘牡丹9’的可溶性糖含量在缺锌与低锌处理下增加幅度并不明显,但在高锌处理下可溶性糖含量迅速增加。低锌和高锌处理都显著降低了‘四单19’叶片的蛋白质含量,而‘牡丹9’低锌处理下叶片可溶性蛋白含量下降并不明显,但在高锌处理下叶片可溶性蛋白含量大幅度下降。低锌处理下,‘四单19’的蛋白质合成受到抑制,而游离氨基酸大量积累,明显抑制了植株的生长;‘牡丹9’的蛋白质和氨基酸含量变化较小。     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini