Castor Bean Seedlings Research Articles

Effects of cadmium and salicylic acid on growth, spectral reflectance and photosynthesis of castor bean seedlings

Salicylic acid (SA) is a potent signaling molecule in plants and is involved in eliciting specific responses to biotic and abiotic stresses. The aim of this study is to investigate whether the exogenous application of SA can improve cadmium (Cd) induced inhibition of photosynthesis in castor bean (Ricinus communis L.) plants. The effects of SA and Cd on plant growth, spectral reflectance, pigment contents, chlorophyll fluorescence and gas exchange were examined in a hydroponic cultivation system. Results indicate that Cd exposure significantly decreased the dry biomass, photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), pigment contents, quantum yield of PS II photochemistry (Fv/Fm), and effective quantum yield of PS II (ΦPS II) in the plants. Pretreatment with SA alone reduced the biomass and Pn in castor bean plants, whereas pigment contents, Fv/Fm and ΦPS II remained unaffected. Reduced Gs, Ci and E, as well as increased stomatal limitation (Ls) and water use efficiency (WUE), were observed in plants pretreated with 500 μM SA alone, whereas plants treated with 250 μM SA were unaffected. Under Cd stress, SA pretreatment led to a significant decrease in Pn, Gs, E, Ci, and chlorophyll contents (Chl a, Chl b, Chl a+b, Car, Chl a/b), and an increase in Ls and WUE. Cd exposure enhanced spectral reflectance in the range 550–680 nm and 750–1,050 nm. It also decreased the normalized difference vegetation index (chlNDI), the modified red edge simple ratio index (mSR705), the red edge position (REP), water band index, and red/green ratio, whereas the structure independent pigment index (SIPI) was increased. Significant correlations (P < 0.01) between spectral indices (mSR705, chlNDI, REP, red/green ratio) and pigment contents. SA significantly worsened plant growth and photosynthesis in Cd-stressed castor bean plants, in which a stomatal limitation was involved. The leaf spectral reflectance is a sensitive indicator in determining Cd toxicity in plants.

The Discovery of Glyoxysomes: the Work of Harry Beevers

Sucrose Synthesis from Acetate in the Germinating Castor Bean: Kinetics and Pathway (Canvin, D. T., and Beevers, H. (1961) J. Biol Chem. 236, 988–995) Mitochondria and Glyoxysomes from Castor Bean Endosperm. Enzyme Constituents and Catalytic Capacity (Cooper, T. G., and Beevers, H. (1969) J. Biol. Chem. 244, 3507–3513)

Leaf gas exchange, chloroplastic pigments and dry matter accumulation in castor bean ( Ricinus communis L) seedlings subjected to salt stress conditions

Brazilian castor bean ( Ricinus communis L) crops have expanded towards semiarid lands, in which soil salinity is an important factor limiting plant development. In order to evaluate the effects of salt stress on leaf gas exchange, pigments, and dry matter accumulation of seedlings, seeds of castor bean var. BRS Nordestina were planted in 15-L pots filled with 13 kg of soil (control) or with soil plus 2 g NaCl kg −1 soil corresponding to 30 mM NaCl (salt treatment). Seedlings were grown under greenhouse conditions and the effects of salinity were assessed by measuring physiological parameters at 38 and 59 days after germination. Salt treatment induced decreases in leaf water potential at pre-dawn ( ψ pd, 42%), stomatal conductance to water vapor ( g s, 36%), and net carbon assimilation rate ( A, 24%) only at 38 days after germination. At the same time, the values for transpiration rate were unchanged and the hydraulic conductance was increased (34%). After 59 days under hypersalinity, leaf ψ pd increased to −0.44 MPa, although g s, A, transpiration rate, and hydraulic conductance were not different between treatments. Salt stress effects on chlorophyll a, chlorophyll b and chlorophyll a + b contents were evident only on experimental day 59. The dry matter accumulation of leaves, roots and stems, as well as the total dry matter, and the root to above ground ratio increased with plant growth in both treatments, however, these parameters were lowered in salt-stressed seedlings. In short, all physiological variables were more drastically affected after 38 days under high salinity, and despite acclimatization of A after 59 days exposure to salt, no recovery of the dry matter accumulation was observed in the seedlings, suggesting that this species does not tolerate salt stress at its initial growth stages.

Dynamics of aquaporins and water relations during hypocotyl elongation in Ricinus communis L. seedlings

The rate of water flow across biological membranes can be modulated by aquaporins which are expressed in many cells and tissues. The biological functions of these water channels in cellular processes have often been anticipated from the expression pattern, although the participation in the underlying process is not known in many cases. Ten putative aquaporin transcripts were identified in castor bean (Ricinus communis L.) seedlings and the water channel activity of three selected genes was analysed by heterologous expression in Xenopus oocytes, as well as the spatial and temporal expression by in situ hybridization/immunolocalization along the hypocotyl's axis. Water relations parameters were studied in elongating and non-elongating tissues using the cell pressure probe technique. These results indicate that (i) the amount of the RcPIP2-1 aquaporin correlated best with the elongation activity of the etiolated hypocotyl and (ii) the hydraulic conductivity of cortex cells is significantly higher in the elongating region of the hypocotyl compared with the non-elongating, mature region.

Phytophthora nicotianae와 P. palmivora var. palmivora에 의한 아주까리 역병

대구시 황금동 주택가에 자라는 아주까리에 발생한 역병(잎마름병)에서 2종의 Phytophthora 균이 분리되었다. 그 하나는 유두돌기가 뚜렷하고, 비탈락성이며, 난형 내지 서양 배 모양의 유주자낭을 형성하고, 그 크기는 31.2-58.5<TEX>${\times}$</TEX>25.4-44.1 <TEX>$\mu\textrm{m}$</TEX>의 범위에 평균 46.4<TEX>$\pm$</TEX>6.5<TEX>${\times}$</TEX>35.9<TEX>$\pm$</TEX>4.7 <TEX>$\mu\textrm{m}$</TEX>, 이주성(Heterothallic)으로서 장란기는 둥글고, 직경 22.5-35.0<TEX>$\mu\textrm{m}$</TEX> 범위에 평균 30<TEX>$\pm$</TEX>3.3<TEX>$\mu\textrm{m}$</TEX>,그 안의 난포자는 충만형이었으며, 직경 18.8-30.0<TEX>$\mu\textrm{m}$</TEX> 범위에 평균 25.4<TEX>$\pm$</TEX>2.8<TEX>$\mu\textrm{m}$</TEX> 이었다. 장정기는 장란기에 저착하였다. 이 종은 Phytophthora nicotianae로 동정되었다. 다른 하나의 종은 유두돌기가 뚜렷하고, 탈락성이며, 난형 내지 타원형, 기부에 자루가 붙어있는 유주자낭을 형성하였으며, 그 크기는 21.5-54.6<TEX>${\times}$</TEX>l7.6-34.3<TEX>$\mu\textrm{m}$</TEX> 범위에 41.7<TEX>$\pm$</TEX>7.1<TEX>${\times}$</TEX>28.4<TEX>$\pm$</TEX>4.0<TEX>$\mu\textrm{m}$</TEX>, 이주성이었으며, 장란기는 구형, 직경 21.3-26.8<TEX>$\mu\textrm{m}$</TEX> 범위에 평균 23.0<TEX>$\pm$</TEX>1.6<TEX>$\mu\textrm{m}$</TEX>, 난포자는 충만형, 직경 17.5-23.8<TEX>$\mu\textrm{m}$</TEX> 범위에 평균 20.2<TEX>$\pm$</TEX>1.8<TEX>$\mu\textrm{m}$</TEX>, 장정기는 저착하였다. 이 종은 P. palmivora (Butler) Butler로 동정되었다. 2종 모두 후막포자를 형성하였다. 이들 2종 Phytophthora 균은 아주까리 유묘의 잎에 강한 병원성을 나타내었다. 이들 2종에 의한 아주까리 역병의 발생은 외국에서는 이미 보고되어 있으나 국내에서는 처음이다. Two species of Phytophthora were isolated from a leaf blight disease found on castor bean plants growing at a residential area of Hwanggeum-dong in Daegu city. One species was producing conspicuously papillate, noncaducous, ovoid to obpyriform sporangia, 31.2-58.5 <TEX>${\times}$</TEX> 25.4-44.1 <TEX>${\mu}{\textrm}{m}$</TEX> in range with an average of 46.4 <TEX>$\pm$</TEX> 6.5 <TEX>${\times}$</TEX> 35.9 <TEX>$\pm$</TEX> 4.7 <TEX>${\mu}{\textrm}{m}$</TEX> on simple sympodial sporangiophores, was heterothallic with oogonia globose, 22.5-35.0 <TEX>${\mu}{\textrm}{m}$</TEX> with an average of 30 <TEX>$\pm$</TEX> 3.3 <TEX>${\mu}{\textrm}{m}$</TEX> in diameter, oospores plerotic, 18.8-30.0 <TEX>${\mu}{\textrm}{m}$</TEX> in range averaging 25.4 <TEX>$\pm$</TEX> 2.8 <TEX>${\mu}{\textrm}{m}$</TEX>, and with antheridia amphigynous. This species was identified as P. nicotianae Breda de Haan. The other species produced papillate, caducous, ovoid to ellipsoid sporangia with pedicel, 21.5-54.6 <TEX>${\times}$</TEX> 17.6-34.3 <TEX>${\mu}{\textrm}{m}$</TEX> in range with an average of 41.7 <TEX>$\pm$</TEX> 7.1 <TEX>${\times}$</TEX> 28.4 <TEX>$\pm$</TEX> 4.0 <TEX>${\mu}{\textrm}{m}$</TEX>, and was heterothallic with oogonia globose, 21.3-26.8 <TEX>${\mu}{\textrm}{m}$</TEX> with an average of 23.0 <TEX>$\pm$</TEX> 1.6 <TEX>${\mu}{\textrm}{m}$</TEX> in diameter, oospores plerotic, 17.5-23.8 <TEX>${\mu}{\textrm}{m}$</TEX> in range averaging 20.2 <TEX>$\pm$</TEX> 1.8 <TEX>${\mu}{\textrm}{m}$</TEX>, and with antheridia amphigynous. This species was identified as P. palmivora (Butler) Butler. Both species produced chlamydospores. Pathogenicity of the 2 speices was confirmed by foliar inoculation of castor bean seedlings. Both species have been reported to infect castor bean plants in a few foreign countries but it is the first that a disease of castor bean plants caused by either or both of the species was found in Korea.

A Metal-binding Member of the Late Embryogenesis Abundant Protein Family Transports Iron in the Phloem ofRicinus communis L.

The transport of metal micronutrients to developing organs in a plant is mediated primarily by the sieve elements. Ligands are thought to form complexes with the free ions in order to prevent cellular damage, but no binding partners have been unequivocally identified from plants so far. This study has used the phloem-mediated transport of micronutrients during the germination of the castor bean seedling to identify an iron transport protein (ITP). It is demonstrated that essentially all (55)Fe fed to seedlings is associated with the protein fraction of phloem exudate. It is shown that ITP carries iron in vivo and binds additional iron in vitro. ITP was purified to homogeneity from minute amounts of phloem exudate using immobilized metal ion affinity chromatography. It preferentially binds to Fe(3+) but not to Fe(2+) and also complexes Cu(2+), Zn(2+), and Mn(2+) in vitro. The corresponding cDNA of ITP was cloned using internal peptide fragments. The deduced protein of 96 amino acids shows high similarity to the stress-related family of late embryogenesis abundant proteins. Its predicted characteristics and its RNA expression pattern are consistent with a function in metal ion binding. The ITP from Ricinus provides the first identified micronutrient binding partner for phloem-mediated long distance transport in plants and is the first member of the late embryogenesis abundant protein family shown to have such a function.

1H Detected 13C Echo Planar Imaging

Cyclic J cross polarisation (CYCLCROP) is a sensitive method for the noninvasive monitoring of 13C distributions and fluxes. The PRAWN rotating frame Hartmann-Hahn mixing sequence ameliorates problems associated with sensitivity to Hartmann-Hahn mismatch and reduces RF power deposition. The combination of CYCLCROP with echo planar imaging (EPI) for spatial encoding of the proton detected carbon signal allows efficient use of the available signal to be made, permitting a significant improvement in the temporal resolution of any study. We report here on some initial experiments to demonstrate the feasibility of echo planar proton detected 13C imaging using CYCLCROP based upon the PRAWN module, including the application of the technique to the measurement of transport and accumulation of 13C-labelled sucrose in a castor bean seedling. Two methods that can be used to eliminate the effect of the J-splitting in the EP images are presented. In addition, a fast, image-based B1 field-mapping method which may be used to quantitatively map the low frequency RF field in a dual resonant (13C/1H) probe is presented. The technique utilises the above described imaging method, permitting fully quantitative, 64×64 axial field maps to be generated in about a minute.

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes.

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.

Characterization of Ricinus communis phloem profilin, RcPRO1.

The mature, functional sieve tube, which forms the conduit for assimilate distribution in higher plants, is dependent upon protein import from the companion cells for maintenance of the phloem long-distance translocation system. Using antibodies raised against proteins present in the sieve-tube exudate of Ricinus communis (castor bean) seedlings, a cDNA was cloned which encoded a putative profilin, termed RcPRO1. Expression and localization studies indicated that RcPRO1 mRNA encodes a phloem profilin, with some expression occurring in epidermal, cortex, pith and xylem tissue. Purified, recombinant RcPRO1 was functionally equivalent to recombinant maize profilin ZmPRO4 in a live cell nuclear displacement assay. The apparent equilibrium dissociation constant for RcPRO1 binding to plant monomeric (G-)actin was lower than the previously characterized maize profilins. Moreover, the affinity of RcPRO1 for poly-L-proline (PLP) was significantly higher than that for recombinant maize profilins. Within the sieve-tube exudate, profilin was present in 15-fold molar excess to actin. The data suggest that actin filament formation is prevented within the assimilate stream. These results are discussed in terms of the unique physiology of the phloem.

Control of gluconeogenesis by isocitrate lyase in endosperm of germinating castor bean seedlings

The aim of this work was to quantify the contribution of isocitrate lyase to the control of gluconeogenesis in endosperm from 4-day-old castor bean seedlings. The approach was based on metabolic control analysis following selective inhibition of enzyme activity. Both 3-nitropropionate and itaconate decreased the proportion of either [1-14C]acetate or [2-14C]acetate converted to sucrose, and increased the proportion metabolized through the tricarboxylic acid cycle. Kinetic analysis of isocitrate lyase activity from endosperm revealed that itaconate is a pure uncompetitive inhibitor (Ki' = 11. 9 +/- 0.98 &mgr;M) with respect to isocitrate. In contrast, 3-nitropropionate is a slow, tight-binding inhibitor. The half-time for inhibition of isocitrate lyase by 3-nitropropionate was 5-10 min, whereas the half-time for reactivation was in excess of 10 h. Incubating endosperm in 3-nitropropionate resulted in a concentration-dependent decrease in isocitrate lyase activity that remained stable in tissue extracts for at least 4 h. From a comparison of the extent of in situ inactivation of isocitrate lyase by 3-nitropropionate and the effect of this compound on the rate of sucrose production from [2-14C]acetate, the flux control coefficient of isocitrate lyase on gluconeogenesis from acetate in castor bean endosperm was calculated to be 0.66 +/- 0.09. It is concluded that isocitrate lyase activity is quantitatively important in the control of gluconeogenic flux, and suggested that developmental changes in the amount of this enzyme may be an important factor in determining the conversion of lipid to sugar in young castor bean seedlings.

Phloem loading and transport characteristics of iron in interaction with plant‐endogenous ligands in castor bean seedlings

The concentration of iron within cells has to be precisely regulated because shortage as well as surplus may be precarious for the survival of the cell. The maintenance of iron homeostasis in shoot organs requires an efficient signalling of the leaf cells’ iron status to the uptake sites of the roots. This ‘iron signal’ may be transferred by the phloem. The handling of iron in the symplast and during phloem transport calls for mechanisms taking into account the specific physicochemical properties of this element.Seedlings of Ricinus communis were used as model plants to investigate characteristic features of phloem loading, and of speciation and valence of iron during transport in the sieve tubes. When the storage endosperm is removed from the cotyledons, phloem is loaded from the reserve pool of the mesophyll cells. In this situation, iron and the other micronutrients copper, manganese and zinc are loaded in a constant stoichiometric ratio of 1:1 to the endogenous complexor nicotianamine (NA). Application of the chelators 1,4‐di(4‐phenylsulphonate)‐1,10‐phenanthroline (BPDS) and ethylenediaminetetraacetic acid to the cotyledon apoplast did not decrease the loading rate of iron, indicating symplastic loading. Supply of ferrous ions in various concentrations to the apoplast revealed the existence of two loading systems. One of them is linearly dependent upon the concentration, and remained unsaturated up to an apoplastic concentration of 200 μM. The other one, whose activity steeply inclines already with a slight increase of the apoplastic concentration, is saturable at a supply of 100 μM. The loading of iron is slowed down with time depending on whether iron is supplied to the apoplast as a complex with NA or citrate instead of as free ferrous ions. This effect may be caused by competition of these chelators with an iron uptake receptor in the plasmalemma of the mesophyll cells. In spite of the close relationship between iron and NA during phloem loading, the Fe‐NA complex seems not to be the predominant transport species in the sieve tubes. A molecule of much larger mass than NA probably serves as a transport vehicle, as concluded from microdialysis experiments. Only 4% of the total iron in the sieve tube exudate was found to exist as Fe(II) and about 45% as Fe(III). The residue of more than 50% was tightly bound and not accessible even in the presence of the reductant sodium dithionite and the chelator BPDS. The conclusions regarding the nature of the transport species and the results on the valence of iron in the sieve tubes were confirmed by calculations with the software programs GEOCHEM [Sposito G, Mattigod SV (1979) A computer program for calculating chemical equilibria in soil solutions and other natural water systems. Kearney Foundation of Soil Sci, Univ. of California, Riverside, CA] and PHREEQC [Parkhurst (1995) PHREEQC‐A computer program for speciation, reaction‐path, adjective‐transport, and inverse geochemical calculations. US Geological Survey]. A model is outlined on the basis of the experimental findings on the fate of iron from mobilisation in the endosperm to trans‐chelation in the sieve tubes.

Investigation of Carbohydrate Metabolism and Transport in Castor Bean Seedlings by CyclicJCross Polarization Imaging and Spectroscopy

NMR experiments using13C-labeled compounds offer the possibility of noninvasive monitoring of carbohydrate transport and metabolism in living plants, but are usually hampered by the low sensitivity of the13C nucleus. The problem of low sensitivity can be overcome by using the cyclicJcross polarization (CYCLCROP) technique, which allows the indirect detection of13C nuclei coupled to1H nuclei with the high NMR sensitivity of protons. We report here on methods for imaging and spectroscopy based on the CYCLCROP technique, and their use in the firstin vivoNMR study of carbohydrate transport and metabolism in castor bean seedlings (Ricinus communis L.). Comprehensive acquisition strategies for the various NMR methods are given, including the procedure for setting up the experiments. In addition, a full analysis of the effect of relaxation on the signals generated from smallJ-coupled spin systems by the CYCLCROP sequence is given, and the high sensitivity of the sequence is demonstrated. In thein vivostudy of six-day-old castor bean seedlings, we were able to measure the uptake of labeled hexoses, supplied in solution to the cotyledons, and their conversion to sucrose, as well as the transport of this sucrose in the vascular bundles. Images of the actual distribution of labeled sucrose in the hypocotyl of the seedling have also been obtained. The resulting data show some evidence for a preferential incorporation of labeled fructose in the process of sucrose synthesis, which decreases with the time of incubation.

Evidence for the Presence of a Porin in the Membrane of Glyoxysomes of Castor Bean.

Glyoxysomes of endosperm tissue of castor bean (Ricinus communis L.) seedlings were solubilized in a detergent and added to a lipid bilayer. Conductivity measurements revealed that the glyoxysomal preparation contained a porin-like channel. Using an electrophysiological method, which we established for semiquantitative determination of porin activity, we were able to demonstrate that glyoxysomal membranes purified by sucrose density gradient centrifugation contain an integral membrane protein with porin activity. The porin of glyoxysomes was shown to have a relatively small single-channel conductance of about 330 picosiemens in 1 M KCl and to be strongly anion selective. Thus, the glyoxysomal porin differs from the other previously characterized porins in the outer membrane of mitochondria or plastids, but is similar to the porin of spinach (Spinacia oleracea L.) leaf peroxisomes. Our results suggest that, in analogy to the porin of leaf peroxisomes, the glyoxysomal porin facilitates the passage of small metabolites, such as succinate, citrate, malate, and aspartate, through the membrane.

A non-invasive measurement of phloem and xylem water flow in castor bean seedlings by nuclear magnetic resonance microimaging

A flow-sensitive nuclear magnetic resonance (NMR) microimaging technique was applied to measure directly the in-vivo water flow in 6-d-old castor bean seedlings. The achieved in-plane resolution of the technique allowed discrimination between xylem and phloem water flow. Both the xylem- and the phloem-average flow velocities in the intact seedling could be quantified. Furthermore, the total conductive cross-sectional area of the xylem vessels and the phloem sieve elements could be determined using the non-invasive and non-destructive NMR microimaging technique. Hence, it was possible to calculate the in-vivo volume flow rates for both xylem and phloem water flow. Our non-destructive technique showed that previously used methods to measure phloem water flow affected the flow rate itself. In the intact seedlings we found values of 16.6 μl·h−1, two fold lower than those previously estimated from phloem exudation rates. Finally, our results demonstrate for the first time that water is internally circulated between phloem and xylem, and that water flow within the xylem is maintained by this internally circulated water, even in the absence of any significant transpiration or evaporation.

High Level Expression ofRicinus communisCasbene Synthase inEscherichia coliand Characterization of the Recombinant Enzyme

Casbene synthase catalyzes the cyclization of geranylgeranyl diphosphate (2) to casbene (1), a diterpene phytoalexin with antibacterial and antifungal activity that is produced by seedlings of castor bean (Ricinus communisL.) in response to fungal attack. We report the high-level expression of casbene synthase cDNA inEscherichia colias insoluble inclusion bodies, the solubilization and refolding of active casbene synthase, and the kinetic and product analysis of the recombinant enzyme. To overcome problems apparently associated with the presence in the casbene synthase gene of rare Arg codons, as well as the intrinsic antibacterial activity of casbene itself, the casbene synthase gene was expressed in anE. colihost harboring the pSM102 vector that encodes thednaYgene fortArg(AGA/G), using an expression vector, pET-21d(+), carrying the tightly controlled T7lacpromoter.

Correlation-Peak Imaging

Identification and quantitation in conventional1H spectroscopic imagingin vivois often hampered by the small chemical-shift range. To improve the spectral resolution of spectroscopic imaging, homonuclear two-dimensional correlation spectroscopy has been combined with phase encoding of the spatial dimensions. From the theoretical description of the coherence-transfer signal in the Fourier-transform domain, a comprehensive acquisition and processing strategy is presented that includes optimization of the width and the position of the acquisition windows, matched filtering of the signal envelope, and graphical presentation of the cross peak of interest. The procedure has been applied to image the spatial distribution of the correlation peaks from specific spin systems in the hypocotyl of castor bean (Ricinus communis) seedlings. Despite the overlap of many resonances, correlation-peak imaging made it possible to observe a number of proton resonances, such as those of sucrose, β-glucose, glutamine/glutamate, lysine, and arginine.

Intracellular Localization of Phospholipase D in Leaves and Seedling Tissues of Castor Bean.

The intracellular distribution of phospholipase D (PLD; EC 3.1.4.4) in castor bean (Ricinus communis L.) tissues was investigated by subcellular fractionation and by immuno-electron microscopy. Centrifugal fractionation revealed that most PLD in young leaves was soluble, whereas in mature leaves a majority of PLD was associated with microsomal membranes. Further separation of microsomal membranes by a two-phase partitioning system indicated that PLD was associated with both plasma and intracellular membranes. Sucrose gradient separation of intracellular membranes showed PLD present in the endoplasmic reticulum, a submicrosomal band, and in soluble fractions but not in mitochondria and glyoxysomes of postgermination endosperm. Immunocytochemical studies found high gold labeling in vacuoles in young leaves, suggesting that the high level of soluble PLD in young leaves is due to release of PLD from vacuoles during tissue disruption. In addition to the labeling in vacuoles, gold particles were also found in the cytoplasmic matrices and plasma membrane in leaves and in 2-d postgermination seedlings. Collectively, these results show that PLD in castor bean leaf and seedling tissues is localized in the vacuole and is associated with the endoplasmic reticulum and plasma membrane and that the relative distribution between the soluble and membrane compartments changes during castor bean leaf development.

Structural Heterogeneity of Phospholipase D in 10 Dicots

The occurrence of multiple forms of phospholipase D (EC 3.1.4.4) was investigated in different tissues of castor bean (Ricinus communis) and in other plant species. Phospholipase D variants were resolved by nondenaturing and isoelectric focusing polyacrylamide gel electrophoresis and detected by immunoblotting using anti-phospholipase D antibodies and by enzyme activity assay. Three phospholipase D variants were produced differentially in the roots, endosperm, cotyledons, and hypocotyl of 5-day postgermination seedlings of castor bean. Furthermore, different patterns of phospholipase D variants were found in the different regions of hypocotyl (elongated and hook). Multiple phospholipase D forms were found in florets of cauliflower and broccoli, leaves of cabbage, celery, tomato, and potato, and alfalfa sprouts, suggesting that structural heterogeneity of phospholipase D occurs widely in plants.

Phloem transport of abscisic acid in Ricinus communis L. seedlings

ABSTRACTSieve tube sap exuded from the cut hypocotyl of castor bean seedlings (Ricinus communis L.) was found to contain 0.2–0.5 mmol m−3abscisic acid (ABA). The ABA concentration in the sieve tube sap always exceeded that in root pressure exudate under a wide range of water supply. Exudation of sieve tube sap from the cut hypocotyls caused water loss, and this induced ‘water shortage’ in the cotyledons which resulted in the ABA concentration in the cotyledons increasing by 3‐fold and that in the sieve tube sap increasing by up to 50‐fold within 7h. The wounded surface of the cut hypocotyl was not responsible for the ABA increase.Incubation of the cotyledons of endosperm‐free seedlings in various ABA concentrations (up to 100 mmol m−3) increased the ABA concentration in sieve tube sap. The concomitant increase in ABA, both in cotyledons and in sieve tube sap, had no effect on the phloem loading of sucrose, K+ and Mg2+ within the experimental period, i.e. up to 10h.It can be concluded that (i) the phloem is an important transport path for ABA, (ii) water stress at the phloem loading sites elevates phloem‐mobile ABA, which may then serve as a water stress signal for sinks, for example stem and roots (not only for stomata), and (iii) the ABA concentration of cells next to or in the phloem is more important than the average ABA content in the whole cotyledon for determining the ABA concentration in sieve tube sap.

Dehydrin-like proteins in castor bean seeds and seedlings are differentially produced in response to ABA and water-deficit-related stresses

The stress inducibility of dehydrin protein production in seedlings of castor bean was analysed by subjecting them to ABA and various water-deficit-related treatments including desiccation, water stress, high salt, high osmolarity, and low temperature. A further goal was to determine whether the immature seed (at stages prior to major dehydrin synthesis) would respond in a similar manner to these stresses. A number of dehydrin-like proteins increased in seedlings subjected to the various stress treatments. In the endosperm, these appear to be different from the dehydrin-related polypeptides that are induced during late seed development and which persist following germination/growth of mature seeds. In the endosperm of seedlings, ABA, water stress and desiccation induced the same dehydrin polypeptides, while high osmolarity, high salt and low temperature induced a different set. Stress-specific differences in dehydrin synthesis were also found in the cotyledons and radicle of castor bean seedlings ; however, dehydrins inducible by exogenous ABA were consistently produced. Immature seeds treated with ABA or subjected to stress responded by producing dehydrin-like proteins associated with late development ; however, the same proteins were induced following detachment of immature seeds from the parent plant and maintenance on water. When seedlings were exposed simultaneously to GA and either ABA, high salt, or low temperature, dehydrin production was suppressed. It is concluded that dehydrin production in castor bean is tissue-specific and is dependent upon the physiological stage of the seed. In the endosperm, the response to different stresses may rely upon more than one signal transduction pathway.