Levels of short-chain fatty acids and of abscisic acid in water-stressed and non-stressed leaves and their effects on stomata in epidermal strips and excised leaves

Straight-chain saturated fatty acids (C6-C11) and abscisic acid (ABA) accumulate in the leaves of Phaseolus vulgaris L. and Hordeum vulgare L. under water stress. ABA and certain of the fatty acids, particularly decanoic and undecanoic acid, can inhibit stomatal opening and cause stomatal closure in epidermal strips of Commelina communis L. depending on the incubating medium used. 10^-4 M (±)-ABA inhibits opening in media containing either high or relatively low concentrations of KCl but causes closure only in the latter medium. The fatty acids (at 10^-4 M) prevent opening in both media while significant closure of open stomata was caused only by undecanoic acid in both media and, additionally, by decanoic acid in the low-KCl medium. 10^-4 M formic acid also caused stomatal closure and prevented opening to significant extents in the low-KCl medium (it was not tested in the high-KCl medium). The efficacy of undecanoic acid in causing 50% inhibition of opening is about three orders of magnitude lower than that of ABA. At a concentration of 10^-3 M, nonanoic, decanoic and particularly undecanoic acid and all-trans-farnesol cause increased cell leakage in Beta vulgaris L. root tissue. Undecanoic acid (10^-4 M) also causes some loss of guard cell integrity in C. communis within 1.5 h of treatment. ABA (10^-4 M) reduces transpiration rates in barley and C. communis leaves when applied via the transpiration stream but decanoic and undecanoic acids did not have this effect. Transpiration was not affected when ABA or the fatty acids were applied to the leaf surfaces.Abbreviations ABA abscisic acid - RWC relative water content - SCFA short-chain fatty acids Deceased May 1977     

Inhibition of stomatal opening in sunflower leaves by carbon monoxide,and reversal of inhibition by light

When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO₂, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm·s^-1. The CO effect on stomatal aperture required a CO/O₂ ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibitio of cytochrome-c oxidase by CO is competitive in respect to O₂. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO₂ was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus.In contrast to red light, very weak blue light (405 nm) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a ₃-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis.     

The effect of Cl- upon the sensitivity of starch-containing and starch-deficient stomata and guard cell protoplasts towards potassium ions,fusicoccin and abscisic acid

Chloride ions are necessary to compensate for the positively charged potassium ions imported into guard cells of Allium cepa L. during stomatal opening. Therefore an external Cl^- supply of intact Allium plants is important. But high levels of chloride have been found to reduce the sensitivity of the starch-lacking stomata and isolated guard cell protoplasts (GCPs) from Allium to potassium ions, fusicoccin and abscisic acid. Furthermore, with high levels of chloride, malate anions disappear from the guard cells of Allium, a finding which contrasts with situation in Vicia where the stomatal sensitivity to K⁺ ions, fusicoccin and ABA is not influenced by Cl^- ions and malate levels are unaffected. It is suggested that the absence of malate as a proton yielding primer inhibits the mechanism of H⁺/K⁺ exchange in Allium.Abbreviations ABA abscisic acid - FC fusicoccin - GCPs guard cell protoplasts     

Short-chained oxygenated VOC emissions in <Emphasis Type="Italic">Pinus halepensis</Emphasis> in response to changes in water availability

Short-chained oxygenated VOC (oxVOCs) emissions from Pinus halepensis saplings were monitored in response to changes in water availability. Online measurements were made with a proton transfer reaction—mass spectrometer under controlled conditions, together with CO₂ and H₂O exchange measurements. Masses corresponding to methanol and acetone were the most emitted oxVOCs. All the oxVOC exchanges, except that of acetone (M59), were significantly related to stomatal conductance and transpiration. Acetaldehyde (M45) emission showed, moreover, a strong dependence on the concentration of acetaldehyde in the ambient: stomatal opening (stomatal conductance above 75 mmol m⁻² s⁻¹) only allowed increased emissions when external concentration were below 6 ppb. Acetone (M59) presented an important peak of emission following light and stomatal opening in the morning when plants were water stressed. Thus, the alterations in oxVOC emissions in P. halepensis caused by the water deficit seem to be mainly driven by water stress effect on stomatal closure and oxVOC air concentrations.     

Isolation and identification of soluble polysaccharides in epidermal tissue of Allium cepa

Because starch is absent from Allium-guard cells, another polysaccharide was sought that, in connection with stomatal opening, could be a source of organic anions. Analysis of isolated polysaccharides revealed xylose, arabinose, glucose, galactose, and galacturonic acid (3.4:1:1.6:0.7) to be components of the water-soluble mucilage of the epidermal strips of Allium cepa. However, the experiments gave no indication that the mucilage is the malate donator during the stomatal opening. After ¹⁴CO₂ fixation the following substances were labeled: organic acids, especially malate and citrate, amino acids and the polysaccharide mentioned above; radioactive 3-phosphoglyceric acid and sugar phosphates were not found. Therefore we conclude that the Calvin cycle does not operate in the guard cells of Allium cepa.Abbreviations ABA Abscisic acid     

Evidence in support of biosynthesis de novo of free cytokinins

The four cytokinins in the tRNA from Lupinus luteus L. seeds have been purified and identified as ribosyl-cis-zeatin, 2-methylthio-ribosylzeatin, ( ²-isopentenyl)adenosine and 2-methylthio-N⁶-( ²-isopentenyl)adenosine. These structures have been assigned on the basis of their chromatographic mobilities and the spectroscopic data of the parent materials and their silylated derivatives. The tRNA isolated from Populus x robusta Schneid. leaves contained four cytokinins with identical chromatographic properties to those identified in Lupinus luteus seed tRNA. No evidence was obtained for the presence, in tRNA, of the naturally occurring free cytokinins identified in these plant species, dihydrozeatin (Lupinus luteus) and N⁶-(2-hydroxybenzyl)adenosine (Populus x robusta). This is evidence in support of the possibility that free cytokinins can arise by biosynthesis de novo and are not exclusively by-products released intact during tRNA turnover.     

Xylem water flow in tropical vines as measured by a steady state heating method

Summary A method for determining the mass flow rate of xylem water in thin stems under natural field conditions is presented. Diurnal courses of xylem water flow and stomatal conductance of the vines Entadopsis polystachya, Cyclanthera multifoliolata, and Serjania brachycarpa were examined in a tropical deciduous forest on the west coast of Mexico. E. polystachya (leaf area 23.6 m²) had a maximum water flow rate of 6.50 kg h^-1 or 1.44 kg cm^-2 _{stem basal area} h^-1; daily water use was 2.00 kg m^-2 _{leaf area} day^-1. S. brachycarpa (leaf area 4.5 m²) and C. multifoliolata (leaf area 3.6 m²) had a maximum water flow rate of 0.72 and 0.19 kg h^-1 or 0.63 and 0.92 kg cm^-2 _{stem basal area} h^-1. Daily water use was 1.26 and 0.39 kg m^-2 _{leaf area} day^-1, respectively. The daily courses of xylem water flow were strongly influenced by the orientation of the leaf area to irradiance and its intensity. While leaves of E. polystachya had a constant high stomatal conductance during the day, S. brachycarpa had a maximum stomatal opening in the morning followed by continuous closure during the rest of the day. In contrast to the woody species, the herbaceous C. multifoliolata exhibited a strong midday depression of stomatal conductance and wilting of its leaves. The leaf biomass accounted for 8% (Entadopsis), 16% (Serjania), and 23% (Cyclanthera) of above-ground biomass. The relation of sapwood area to leaf area supplied (Huber value) was 0.19 (Entadopsis), 0.18 (Serjania), and 0.06 (Cyclanthera) cm² m^-2     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Promotion of stomatal opening in the grass Anthephora pubescens nees by a range of natural and synthetic cytokinis

Stomatal opening in detached epidermis of Anthephora pubescens Nees incubated in the light and CO₂-free air was enhanced by each of 6 natural and 4 synthetic cytokinis. Apertures were maximal following incubation with 10 mmol m^-3 cytokinin in PIPES buffer for all except N⁶-[ ²-isopentyl] adenine and N⁶-[ ²-sopentyl] adenosine which both opened stomata maximally at 0.1 mmol m^-3. Experiments which were undertaken to optimise the conditions of incubation showed that opening was maximal after 3 h incubation and that while 10 mmol m^-3 kinetin increased the rate of stomatal opening, it did not affect the duration. Exogenous KCl was not needed for opening and light was saturating even at the low level of 140 mol m^-2 s^-1.     

Vanadate inhibition of stomatal opening in epidermal peels of Commelina communis

An H⁺ ATPase at the plasma-membrane of guard cells is thought to establish an electrochemical gradient that drives K⁺ and Cl^– uptake, resulting in osmotic swelling of the guard cells and stomatal opening. There are, however, conflicting results regarding the effectiveness of the plasma-membrane H⁺-ATPase inhibitor, vanadate, in inhibiting both H⁺ extrusion from guard cells and stomatal opening. We found that 1 mM vanadate inhibited light-stimulated stomatal opening in epidermal peels of Commelina communis L. only at KCl concentrations lower than 50 mM. When impermeant n-methylglucamine and HCl (pH 7.2) were substituted for KCl, vanadate inhibition was still not observed at total salt concentrations50 mM. In contrast, in the absence of Cl^–, when V₂O₅ was used to buffer KOH, vanadate inhibition of stomatal opening occurred at K⁺ concentrations as high as 70 mM. Partial vanadate inhibition was observed in the presence of the impermeant anion, iminodiacetic acid (100 mM KHN(CH₂CO₂H)₂). These results indicate that high concentrations of permeant anions prevent vanadate uptake and consequently prevent its inhibitory effect. In support of this hypothesis, an inhibitor of anion uptake, anthracene-9-carboxylic acid, partially prevented vanadate inhibition of stomatal opening. Other anion-uptake inhibitors (1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 1 mM 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid, 200 M Zn²⁺) were not effective. Decreased vanadate inhibition at high Cl^–/vanadate ratios may result from competition between vanadate and Cl^– for uptake. Unlike metabolic inhibitors, vanadate did not affect the extent of stomatal closure stimulated by darkness, further indicating that the observed action of vanadate represents a specific inhibition of the guard-cell H⁺ ATPase.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - FC fusicoccin - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid We thank Drs. R.T. Leonard (University of California, Riverside, USA) and K.A, Rubinson (Yellow Springs, Oh., USA) for helpful comments on the research, Janet Sherwood (Harvard University) for excellent plant care, and Angela Ciamarra, Anne Gershenson, Gustavo Lara (Harvard University) and Orit Tal (Hebrew University) for valuable technical assistance. This research was supported by a grant from the National Science Foundation (DCB-8904041) to S.M.A.     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Slow photosynthetic induction and low photosynthesis in Paphiopedilum armeniacum are related to its lack of guard cell chloroplast and peculiar stomatal anatomy

Paphiopedilum and Cypripedium are close relatives in the subfamily Cypripedioideae. Cypripedium leaves contain guard cell chloroplasts, whereas Paphiopedilum do not. It is unclear whether the lack of guard cell chloroplasts affects photosynthetic induction, which is important for understory plants to utilize sunflecks. To understand the role of guard cell chloroplasts in photosynthetic induction of Paphiopedilum and Cypripedium, the stomatal anatomy and photosynthetic induction of Paphiopedilum armeniacum and Cypripedium flavum were investigated at different ratios of red to blue light. The highest stomatal opening and photosynthesis of intact leaves in P. armeniacum were induced by irradiance enriched with blue light. Its stomatal opening could be induced by red light 250 µmol m^?2 s^?1, but the magnitude of stomatal opening was lower than those at the other light qualities. However, the stomatal opening and photosynthesis of C. flavum were highly induced by mixed blue and red light rather than pure blue or red light. The two orchid species did not differ in stomatal density, but P. armeniacum had smaller stomatal size than C. flavum. The stomata of P. armeniacum were slightly sunken into the leaf epidermis, while C. flavum protruded above the leaf surface. The slower photosynthetic induction and lower photosynthetic rate of P. armeniacum than C. flavum were linked to the lack of guard cell chloroplasts and specific stomatal structure, which reflected an adaptation of Paphiopedilum to periodic water deficiency in limestone habitats. These results provide evidence for the morphological and physiological evolution of stomata relation for water conservation under natural selection.     

Seasonal changes of cytokinins in upper and lower leaves of a sugar maple crown

Although it is well accepted that cytokinins (CKs) regulate processes such as leaf senescence and stomatal conductance, data on CKs in the canopy of mature trees are lacking in the literature. Here we report the first in situ sampling for determination of CKs in mature sugar maple (Acer saccharum) canopy layers. The upper canopy showed a distinct seasonal pattern in total CK content, while the lower canopy remained relatively unchanged.This project was supported by an NSERC Discovery Grant to RJNE.     

Nutrient and water addition effects on day- and night-time conductance and transpiration in a C3 desert annual

Recent research has shown that many C₃ plant species have significant stomatal opening and transpire water at night even in desert habitats. Day-time stomatal regulation is expected to maximize carbon gain and prevent runaway cavitation, but little is known about the effect of soil resource availability on night-time stomatal conductance (g) and transpiration (E). Water (low and high) and nutrients (low and high) were applied factorially during the growing season to naturally occurring seedlings of the annual Helianthus anomalus. Plant height and biomass were greatest in the treatment where both water and nutrients were added, confirming resource limitations in this habitat. Plants from all treatments showed significant night-time g (~0.07 mol m⁻² s⁻¹) and E (~1.5 mol m⁻² s⁻¹). In July, water and nutrient additions had few effects on day- or night-time gas exchange. In August, however, plants in the nutrient addition treatments had lower day-time photosynthesis, g and E, paralleled by lower night-time g and E. Lower predawn water potentials and higher integrated photosynthetic water-use efficiency suggests that the nutrient addition indirectly induced a mild water stress. Thus, soil resources can affect night-time g and E in a manner parallel to day-time, although additional factors may also be involved.     

Alterations in Rubisco activity and in stomatal behavior induce a daily rhythm in photosynthesis of aerial leaves in the amphibious-plant Nuphar lutea

Nuphar lutea is an amphibious plant with submerged and aerial foliage, which raises the question how do both leaf types perform photosynthetically in two different environments. We found that the aerial leaves function like terrestrial sun-leaves in that their photosynthetic capability was high and saturated under high irradiance (ca. 1,500 μmol photons m⁻² s⁻¹). We show that stomatal opening and Rubisco activity in these leaves co-limited photosynthesis at saturating irradiance fluctuating in a daily rhythm. In the morning, sunlight stimulated stomatal opening, Rubisco synthesis, and the neutralization of a night-accumulated Rubisco inhibitor. Consequently, the light-saturated quantum efficiency and rate of photosynthesis increased 10-fold by midday. During the afternoon, gradual closure of the stomata and a decrease in Rubisco content reduced the light-saturated photosynthetic rate. However, at limited irradiance, stomatal behavior and Rubisco content had only a marginal effect on the photosynthetic rate, which did not change during the day. In contrast to the aerial leaves, the photosynthesis rate of the submerged leaves, adapted to a shaded environment, was saturated under lower irradiance. The light-saturated quantum efficiency of these leaves was much lower and did not change during the day. Due to their low photosynthetic affinity for CO₂ (35 μM) and inability to utilize other inorganic carbon species, their photosynthetic rate at air-equilibrated water was CO₂-limited. These results reveal differences in the photosynthetic performance of the two types of Nuphar leaves and unravel how photosynthetic daily rhythm in the aerial leaves is controlled.     

Periodic Nocturnal Stomatal Opening of Citrus in a Steady Environment

In a steady environment, leaf stomates of sour orange (Citrus aurantium L.) remained closed in the dark, whereas those of rough lemon (Citrus jambhiri Lush) underwent several 20-minute periods of pronounced opening at intervals of 60–85 minutes. Peak nocturnal opening occurred about 10 minutes after the cycle began, as shown by a change in leaf diffusion resistance from 60 s cm^?1 (closed) to 2 s cm^minus;1 (open). This minimal leaf resistance equals that obtainable in rough lemon in strong illumination (26 kilolux). The transitory stomatal opening detected by leaf resistance measurements was corroborated by measurements of leaf temperature and leaf thickness. During nocturnal opening, leaf temperature was 2°C below the value representing non-cycling periods. Also, minimal leaf thickness coincided with maximal stomatal opening. The triggering mechanism for nocturnal stomatal opening appears to originate within the plant, since the environmental factors of air temperature, humidity, carbon dioxide concentration, and substrate (aerated water culture) were held steady.     

The effect of wavelength of light on stomatal opening

The effect of broad band green, blue and red light on stomatal opening of Vicia faba L. (broad bean) leaves was examined. In air, blue light caused greater stomatal opening than red light. In air with green light stomata were only slightly opened. In a nitrogen atmosphere red light caused greater opening than blue light, and green light caused only slight opening. Opening in air or nitrogen atmosphere in red or blue light was inhibited by the uncoupler CCCP, while the photosynthetic inhibitor DCMU inhibited opening in air but not in nitrogen atmosphere. We concluded that more than one light activated metabolic pathway can supply the energy needed to effect stomatal opening and that different pathways are operative under different conditions.     

Metabolic energy for stomatal opening. Roles of photophosphorylation and oxidative phosphorylation

The supply of energy for stomatal opening was investigated with epidermal peels of Commelina communis L. and Vicia faba L., under white, blue and red irradiation or in darkness. Fluencerate response curves of stomatal opening under blue and red light were consistent with the operation of two photosystems, one dependent on photosynthetic active radiation (PAR) and the other on blue light, in the guard cells. The PAR-dependent system was 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-sensitive and KCN-resistant and showed a relatively high threshold irradiance for its activation; its activity was most prominent at moderate to high irradiances. The blue-light-dependent photosystem was KCN-sensitive, was active at low irradiances, and interacted with the PAR-dependent photosystem at high blue irradiances. Stomatal opening in darkness, caused by CO₂-free air, fusicoccin or high KCl concentrations, was KCN-sensitive and DCMU-resistant. These data indicate that stomatal opening in darkness depends on oxidative phosphorylation for the supply of high-energy equivalents driving proton extrusion. Light-dependent stomatal opening appears to require photophosphorylation from guard-cell chloroplasts and the activation of the blue-light photosystem which could rely either on oxidative phosphorylation or a specific, membrane-bound electron-transport carrier.Abbreviations DCMU 3(3,4-dichlorophenyl)-1-1-dimethylurea - FC fusicoccin - KCN potassium cyanide - PAR photosynthetic active radiation - WL white light     

Spatial and temporal changes in endogenous cytokinins in developing pea roots

Germination and seedling establishment follows a distinct pattern which is partly controlled by hormones. Roots have high levels of cytokinins. By quantifying the fluctuations in endogenous cytokinins over time, further insight may be gained into the role of cytokinins during germination and seedling establishment. Radicles were excised from sterile Pisum sativum L. seeds after 30 min and 5 h imbibition. Seedlings germinated on agar were harvested after 1, 3, 6 and 9 days. The roots were divided into the root tip, root free zone, secondary root zone and from day 6, the secondary roots. Samples were purified by various chromatographic methods and endogenous cytokinins detected by LC(+)ES-MS. Benzyladenine levels doubled after 5 h imbibition and then gradually decreased over time. Low concentrations of cis-Zeatin (cZ) type cytokinins were detected in the radicle after 30 min imbibition. After 5 h imbibition, cis-zeatin riboside-5′-monophosphate had greatly increased. The total cytokinin content of the roots increased over time with the ribotides being the predominant conjugates. From day 3 onwards, there was a gradual increase in the free bases, O-glucosides and their ribosylated forms. Mainly N ⁶ -(2-isopentenyl)adenine (iP)-type cytokinins were detected in the root tip, whereas trans-zeatin- (tZ), dihyrozeatin- (DHZ) and iP-type cytokinins were found in the secondary roots and root zone. Cytokinin biosynthesis was only detected after day 6. Biosynthesis of iP and tZ derivatives was quite rapid, whereas biosynthesis of cZ derivatives remained at a low basal level. These fluctuations in cytokinin types and concentrations suggest the cytokinins may be synthesized from various pathways in pea roots.