Subcellular localization of a high affinity binding site for D-myo-inositol 1,4,5-trisphosphate from Chenopodium rubrum.

It is now generally accepted that a phosphoinositide cycle is involved in the transduction of a variety of signals in plant cells. In animal cells, the binding of D-myo-inositol 1,4,5-trisphosphate (InsP(3)) to a receptor located on the endoplasmic reticulum (ER) triggers an efflux of calcium release from the ER. Sites that bind InsP(3) with high affinity and specificity have also been described in plant cells, but their precise intracellular locations have not been conclusively identified. In contrast to animal cells, it has been suggested that in plants the vacuole is the major intracellular store of calcium involved in signal induced calcium release. The aim of this work was to determine the intracellular localization of InsP(3)-binding sites obtained from 3-week-old Chenopodium rubrum leaves. Microsomal membranes were fractionated by sucrose density gradient centrifugation in the presence and absence of Mg(2+) and alternatively by free-flow electrophoresis. An ER-enriched fraction was also prepared. The following enzymes were employed as specific membrane markers: antimycin A-insensitive NADH-cytochrome c reductase for ER, cytochrome c oxidase for mitochondrial membrane, pyrophosphatase for tonoplast, and 1,3-beta-D-glucansynthase for plasma membrane. In all membrane separations, InsP(3)-binding sites were concentrated in the fractions that were enriched with ER membranes. These data clearly demonstrate that the previously characterized InsP(3)-binding site from C. rubrum is localized on the ER. This finding supports previous suggestions of an alternative non-vacuolar InsP(3)-sensitive calcium store in plant cells.

Cytokinins in tobacco and wheat chloroplasts. Occurrence and changes due to light/dark treatment.

Although cytokinins (CKs) affect a number of processes connected with chloroplasts, it has never been rigorously proven that chloroplasts contain CKs. We isolated intact chloroplasts from tobacco (Nicotiana tabacum L. cv SR1) and wheat (Triticum aestivum L. cv Ritmo) leaves and determined their CKs by liquid chromatography/tandem mass spectroscopy. Chloroplasts from both species contained a whole spectrum of CKs, including free bases (zeatin and isopentenyladenine), ribosides (zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5'-monophosphate, zeatin riboside-5'-monophosphate, and dihydrozeatin riboside-5'-monophosphate), and N-glucosides (zeatin-N(9)-glucoside, dihydrozeatin-N(9)-glucoside, zeatin-N(7)-glucoside, and isopentenyladenine-N-glucosides). In chloroplasts there was a moderately higher relative amount of bases, ribosides, and ribotides than in leaves, and a significantly increased level of N(9)-glucosides of zeatin and dihydrozeatin. Tobacco and wheat chloroplasts were prepared from leaves at the end of either a dark or light period. After a dark period, chloroplasts accumulated more CKs than after a light period. The differences were moderate for free bases and ribosides, but highly significant for glucosides. Tobacco chloroplasts from dark-treated leaves contained zeatin riboside-O-glucoside and dihydrozeatin riboside-O-glucoside, as well as a relatively high CK oxidase activity. These data show that chloroplasts contain a whole spectrum of CKs and the enzymatic activity necessary for their metabolism.

Diurnal Fluctuations in Ethylene Formation in Chenopodium rubrum.

Ethylene formation was studied in 5- to 6-d-old Chenopodium rubrum seedlings under the following light regimes: continuous light (CL), continuous darkness (CD), and alternating light/darkness (12 h of each). No significant regular oscillations in ethylene formation were found in either the CL or CD groups. In the light/dark regime, pronounced diurnal fluctuations in ethylene formation were observed. Activity of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase was transiently increased on transfer from light to dark and vice versa. In CL, ACC oxidase activity did not change significantly, whereas in CD, it decreased continuously after the initial increase. The in vivo levels of ACC and N-malonyl-ACC (MACC) were constant for the first few hours of darkness, then decreased dramatically, but increased again in the light. In constant darkness, the level of ACC displayed endogenous rhythm, with minimum values at h 12 and 44, and a maximum value at h 32 to 36. The level of MACC in both shoots and roots decreased in the CD group until h 12, and then remained constant until h 30 before decreasing continuously. We conclude that the photoperiodic regime affects both ACC and MACC levels, as well as the conversion of ACC to ethylene. Correlation of the described changes in ethylene formation to photoperiodic flower induction is discussed.

Identification and Preliminary Characterization of a Ca2+- Dependent High-Affinity Binding Site for Inositol-1,4,5-Trisphosphate from Chenopodium rubrum.

Using a radioligand-binding assay we have identified a Ca2+- dependent high-affinity D-myo-inositol-1,4,5-trisphosphate (InsP3) binding site in a membrane vesicle preparation from Chenopodium rubrum. Millimolar concentrations of Ca2+ were required to observe specific binding of [3H]InsP3. A stable equilibrium between bound and free ligand was established within 5 min and bound [3H]InsP3 could be completely displaced by InsP3 in a time- and concentration-dependent manner. Displacement assays indicated a single class of binding sites with an estimated dissociation constant of 142 [plus or minus] 17 nM. Other inositol phosphates bound to the receptor with much lower affinity. The glycosaminoglycan heparin was an effective competitor for the binding site (inhibitor concentration for 50% displacement = 534 nM). ATP at higher, although physiologically relevant, concentrations (inhibitor concentration for 50% displacement = 241 [mu]M) also displaced [3H]InsP3 from the receptor. Recent studies in animals have highlighted the importance of Ca2+ regulation of InsP3-induced Ca2+ release. The potential for the operation of similar regulatory mechanisms in plants is discussed.

Effects of agrobacterial oncogenes in kidney vetch (Anthyllis vulneraria L.).

Kidney vetch seedlings were induced to form hairy roots by inoculating their mesocotyls with the wild-type strain 15834 of Agrobacterium rhizogenes or with the A. tumefaciens strain C58C1 containing a binary vector system (the pRiA4b as a helper and the vector pCB1346 bearing a pTiC58-derived isopentenyl transferase gene (ipt, cytokinin biosynthetic gene) under control of its native regulatory sequences). Transgenic lines of three distinct phenotypes were selected: (i) Typically, the pRi15834-transformed tissues were stabilized in vitro and maintained for long periods as aseptic, fast-growing, hormone-independent, plagiotropic hairy root cultures which never regenerated shoots and lost the ability to synthesize opines. Their genomic DNA contained both the TL- and the TR-DNA. (ii) One of the HR-lines transgenic for the T-DNA of pRi15834 (named 52AV34) started to regenerate spontaneously into teratomous shoots. The shoots were found to produce opines and both the TL and TR parts of T-DNA were found to be partly deleted and/or rearranged. They contained phytohormones in similar levels as those found in seed-born shoots. (iii) A practically identical morphogenic response as in the line 52AV34 was observed in the clone 27AV46. However, its shooty, dark-green, slow-growing teratomas were proven to be kanamycin-resistant, opine-producing, and double-transformed by the pRiA4b sequences and the ipt gene. They over-produced auxins as well as cytokinins (mainly indoleacetylaspartic acid and ribosides of zeatin and isopentenyladenine).

Phenylalanine ammonia-lyase, phenolic acids and ethylene in alfalfa (Medicago sativa L.) cell cultures in relation to their embryogenic ability.

Phenylalanine ammonia-lyase (PAL) activity, contents of phenolic acids and ethylene production during the lag-phase, and contents of phenolic acids at the late exponential phase, showed significant differences in embryogenic (EC) and non-embryogenic (NEC) suspension cultures of Medicago sativa L. Maximum PAL activity at 6 h after inoculation was followed by an increase in the level of phenolic acids from 9.6 µg g(-1) fresh mass to 21 µg g(-1) fresh mass in NEC at 12 h. Thereafter the level of phenolic acids decreased to 5.2 µg g(-1) fresh mass at 72 h. The decline was caused predominantly by the decrease of ester-bound cinnamic acid derivatives, the decrease ranging from 83 to 20% of total phenolics. Two maxima of ethylene production were observed in NEC: the first one immediately after inoculation and the second at 6 h, coinciding with the peak of PAL activity. In NEC, most of the phenolic acids occurred in esterified form. Ability to form somatic embryos (EC) was associated with the absence of the second peak of ethylene production as well as of the peak of PAL activity at 6 h. The level of phenolic acids during the lag-phase remained low (7.2 µg g(-1) FM) and did not change. The proportion of cinnamic acid derivatives was very low (18% of total phenolics), mostly due to the extremely low level of ferulic acid. In EC, phenolic acids bound to methanol insoluble material formed the major fraction. Loss of embryogenic potential of the embryogenic culture (ECL) was associated with qualitative and quantitative changes in the contents of phenolic acids insignificantly increased PAL activity after inoculation was followed by a moderate increase in the contents of phenolic acids from 9.35 µg g(-1) fresh mass to 12.42 µg g fresh mass. A high rate of ethylene production was observed only immediately after the transfer of the culture to fresh medium. The loss of embryogenicity correlated also with changes in the relative amounts of the investigated fractions of phenolic acids. A distinct increase in the level of methoxy-substituted phenolic acids is a characteristic feature of the ECL culture.