Growth and gibberellin-A1 metabolism in normal and gibberellin-insensitive (Rht3) wheat (Triticum aestivum L.) seedlings

Growth parameters were determined for tall (rht3) and dwarf (Rht3) seedlings of wheat (Triticum aestivum L.). Plant statures and leaf length were reduced by 50% in dwarfs but root and shoot dry weights were less affected. Leaves of dwarf seedlings had shorter epidermal cells and the numbers of cells per rank in talls and dwarfs matched the observed relationships in overall length. Talls grew at twice the rate of dwarfs (2.3 compared with 1.2 mm h^-1). [³H]Gibberellin A₁ ([³H]GA₁) was fed to seedlings via the third leaf and metabolism was followed over 12 h. Immature leaves of tall seedlings transferred radioactivity rapidly to compounds co-chromatographing with [³H]gibberellin A₈ ([³H]GA₈) and a conjugate of [³H]GA₈, whereas leaves of dwarf seedlings metabolised [³H]GA₁ more slowly. Roots of both genotypes produced [³H]GA₈-like material at similar rates. Isotopic dilution studies indicated a reduced 2-hydroxylation capacity in dwarfs, but parallel estimates of the endogenous GA pool size, obtained by radioimmunoassay, indicated a 12–15 times higher level of GA in the dwarf immature leaves. Dwarfing by the Rht3 gene does not appear to operate through enhanced, or abnormal metabolism of active gibberellins and the act of GA metabolism does not bear an obligate relationship to the growth response.Abbreviations GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

The membranes of slowly drought-stressed wheat seedlings: a freeze-fracture study

Seedlings of Triticum aestivum L. cv. Neepawa were slowly drought-stressed by witholding water after sowing in pots. Leaf extension stopped during development of the third leaf. Damage was assessed by rewatering the pots and measuring regrowth; 1–5 d after growth stopped, rewatering induced significant regrowth within several hours; 6–13 d after growth stopped, regrowth was delayed; from 14 d after growth stopped, no regrowth occurred after rewatering. Leaf bases were excised from the drought-stressed seedlings during this period of increasing damage, and were freeze-etched.Intramembranous particles (IMP) were evenly scattered in the plasma membrane in those plants which regrew immediately after rewatering. In the plants which regrew after a delay or which did not regrow on rewatering, there were patches without IMP in plasma membrane, nuclear envelope, and other membranes. Plasma membrane, nuclear envelope and possibly other membranes were sometimes partly replaced by vesicles, possibly formed from the original membrane. Such vesiculation occurred in a few cells in plants which survived the stress with a delayed regrowth, and was commoner in the plants which did not recover. The results support the idea that slow drought induces IMP-free patches in membranes including the plasma membrane, this induces membrane reorganisation including vesiculation of membranes and coagulation of protoplasm, and that these are expressed as delayed or failed regrowth. Some IMP-free patches in the plasma membrane had a faint ordered sub-structure, possibly a hexagonal lipid phase. Such patches were infrequent and IMP sometimes occurred in areas of plasma membrane having an apparently similar sub-structure. Thus the IMP-free patches could not be explained by a lamellar-hexagonal phase transition. As the stress became damaging, vesicles and endoplasmic reticulum accumulated immediately next to the plasma membrane. Mainly during the early period of damaging stress (6–10 d after growth stopped), depressions, invaginations, and rarer lesions occurred in the plasma membrane, sometimes associated with some of the IMP-free patches. In the same period, many nuclear envelopes had exceptionally large nuclear pores.Abbreviations E exoplasmic - IMP intramembranous particles - P protoplasmic     

Changes in the content of modified nucleotides of total transfer RNA of wheat seedlings during greening

The contents in minor nucleotides of total transfer RNA (tRNA) of etiolated and light-grown wheat (Triticum aestivum L.) seedlings and of seedlings illuminated for 24 or 48 h were examined. The total tRNA of seedlings illuminated 24 h contained more, and that from seedlings illuminated 48 h still more modified nucleotides than that from etiolated ones. Thus, the appearance of the characteristic minor nucleotides of tRNA of light-grown wheat seedlings needs a rather long greening period, of at least 48 h.     

The integration of whole-root and cellular hydraulic conductivities in cereal roots

The hydraulic conductivities of excised whole root systems of wheat (Triticum aestivum L. cv. Atou) and of single excised roots of wheat and maize (Zea mays L. cv. Passat) were measured using an osmotically induced back-flow technique. Ninety minutes after excision the values for single excised roots ranged from 1.6·10^-8 to 5.5·10^-8 m·s^-1·MPa^-1 in wheat and from 0.9·10^-8 to 4.8·10^-8 m·s^-1·MPa^-1 in maize. The main source of variation was a decrease in the value as root length increased. The hydraulic conductivities of whole root systems, but not of single excised roots, were smaller 15 h after excision. This was not caused by occlusion of the xylem at the cut end of the coleoptile. The hydraulic conductivities of epidermal, cortical and endodermal cells were measured using a pressure probe. Epidermal and cortical cells of both wheat and maize roots gave mean values of 1.2·10^-7 m·s^-1·MPa^-1 but in endodermal cells (measured only in wheat) the mean value was 0.5·10^-7 m·s^-1·MPa^-1. The cellular hydraulic conductivities were used to calculate the root hydraulic conductivities expected if water flow across the root was via transcellular (vacuole-to-vacuole), apoplasmic or symplasmic pathways. The results indicate that, in freshly excised roots, the bulk of water flow is unlikely to be via the transcellular pathway. This is in contrast to our previous conclusion (H. Jones, A.D. Tomos, R.A. Leigh and R.G. Wyn Jones 1983, Planta 158, 230–236) which was based on results obtained with whole root systems of wheat measured 14–15 h after excision and which probably gave artefactually low values for root hydraulic conductivity. It is now concluded that, near the root tip, water flow could be through a symplasmic pathway in which the only substantial resistances to water flow are provided by the outer epidermal and the inner endodermal plasma membranes. Further from the tip, the measured hydraulic conductivities of the roots are consistent with flow either through the symplasmic or apoplasmic pathways.Symbols L _{p, cell} cell hydraulic conductivity - L _{p, root} root hydraulic conductivity - L _{p, root} calculated root hydraulic conductivity - root reflection coefficient     

Responses of wheat plants to nutrient deprivation may involve the regulation of water-channel function

The sap flow (Jv) and the osmotic hydraulic conductance (L₀) of detached, exuding root systems from wheat (Triticum aestivum L. cv. Chinese Spring) plants deprived of nitrogen for 5 d (— N) or of phosphorus for 7 d (—P), were measured and compared with controls receiving a complete nutrient supply. In the roots of — N and — P plants, Jv and L₀ decreased markedly, but between 4 and 24 h after resupplying N to — N plants (NRS plants) and P to — P plants (PRS plants), Jv and Lo recovered to values similar to those of control plants. Values of Jv and L₀ were always greater during the light period than during the dark, due to the diurnal variation of these parameters. Reducing transpiration in the light had no effect on Jv and L₀ of — N and — P plants. Sap flow and L₀ were also determined using individual axes from plants which had been grown with their roots divided between nutrient-deficient (- N or- P) solution and a complete nutrient solution. Differences were observed in Jv and L₀ between axes of the same plant, but stomatal conductance (Gs), which was also measured, was not affected in these split-root experiments. In control plants, Jv and L₀ declined sharply to values similar to those of roots from — N and — P plants after HgCl₂ treatment (50 M), but were restored by treating with 5 mM dithiothreitol. In plasma membranes from — N and — P roots, the amount of stigmasterol increased relative to sitosterol compared with control roots. The degree of unsaturation of bound fatty acids also increased, compared with controls, as a result of a decline in the relative amounts of 160 and 180 and an increase in 182. Plasma-membrane fluidity, estimated by steady-state fluorescence polarisation using 1,6-diphenyl hexatriene, showed that the plasma membranes from nutrient-deprived plants were less fluid than those from control plants, measured during both the light and dark periods and in split-root experiments. In NRS plants, the relative abundance of sitosterol increased, so that the stigmasterol/sitosterol ratio returned to a value similar to that of controls. However, in PRS plants, the difference in stigmasterol/sitosterol ratio was maintained, compared with controls. The degree of unsaturation of bound fatty acids, membrane fluidity and the hydraulic conductivity of root systems also recovered in NRS and PRS plants to values similar to those of control plant plasma membranes. The results obtained suggested that — N and — P treatment decreased L₀, by reducing either the activity or the abundance of Hg-sensitive water channels. Also, there may be an interaction between the increase in membrane lipid ordering and the decrease in L₀.Abbreviations DTT dithiothreitol - Gs stomatal conductance - Js solute flow into the sap - Jv sap flow - L₀ hydraulic conductance - - N nitrogen-deprived for 5 d - NRS nitrogen resupplied after 5 d deprivation - - P phosphorus-deprived for 7 d - PRS phosphorus resupplied after 7 d deprivation M.C. was funded by a grant from CSIC, Spain. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.     

Only xylem-borne factors can account for systemic wound signalling in the tomato plant

Rapid and systemic defence responses occur in various higher plants, including the tomato (Lycopersicon esculentum L.). The long-distance signalling mechanisms which permit these responses are not clear, but three models are currently considered in the literature: phloem transport, hydraulic dispersal in the xylem, and electrical transmission. Experiments presented here are designed to discriminate between these three models on the basis of some key predictions. It is demonstrated that wound signalling can be prevented by enclosure of the shoots in polythene bags, to generate high humidity. This effect can be reversed by addition of mannitol solution to the roots, showing that it depends on saturation of the plant's water status rather than on changes in the gaseous environment of the shoot. In addition, wound signals are shown to pass freely across heat-killed tissue. These results are predicted by the hydraulic-dispersal model of signalling, but they are not compatible with the other two models. We therefore conclude in favour of hydraulic dispersal.Abbreviations PI proteinase inhibitor - PIIF proteinase-inhibitor-inducing factor This work was supported by the Agriculture and Food Research Council (UK). J.-J.A. is grateful for a Fellowship from the Ministerio de Education y Ciencia (Spain).     

Effect of light on the nucleotide composition of rRNA of wheat seedlings

Both qualitative and quantitative differences in the minor nucleotide constituents of rRNA from normally grown and from etiolated wheat plants (Triticum aestivum L.) were established. Using different degradation methods and separation techniques the 18S+26S RNA of 8-day-old wheat seedlings grown in the light was found to contain 5-methylcytidine, 3-methylcytidine, 5-methyluridine, 3-methyluridine, 5-carboxymethyluridine, 1-methyladenine, N-methyladenine, 5-hydroxymethylcytidine, O²-methyluridine, O²-methylcytidine, pseudouridine, O²-methylpseudouridine, N²,N²-dimethylguanine, 1-methylguanine, ribothymidine and some unknown minor constituents. On the other hand, there were only a few minor nucleotides in the rRNA of etiolated wheat seedlings. Cycloheximide, a cytoplasmic protein synthesis inhibitor, simulated etiolation in that it reduced the number of minor nucleotides in rRNA, whereas chloramphenicol, a chloroplast protein synthesis inhibitor, had no significant effect on the minor nucleotide content of rRNA. This finding suggests that illumination may cause de novo synthesis of cytoplasmic modifying enzymes leading to the formation of highly modified rRNAs.Abbreviations m⁶A N⁶-methyladenine - m¹A 1-methyladenine - 5hmc 5-hydroxymethylcytidine - Cm O²-methylcytidine - m⁵C 5-methylcytidine - m³C 3-methylcytidine - m¹G 1-methylguanine - m ₂ ² G N², N²-dimethylguanine - pseudouridine - m O²-methylpseudouridine - Um O²-methyluridine - m³U 3-methyluridine - m⁵U 5-methyluridine - cm⁵U 5-carboxymethyluridine - rT ribothymidine - Pur purine - Pyr pyrimidine - RNase ribonuclease - UV ultra violet - p phosphate     

ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Purification and characterization of phosphoribulokinase from wheat leaves

Homogeneous phosphoribulokinase (PRK; ATP: d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was isolated from wheat leaves with a specific activity of 15 kat mg^-1 protein. The purification included ammonium sulfate cuts, isoelectric precipitation, and hydrophobic and affinity chromatography on pentylagarose and Blue Sepharose CL 6B, respectively. Gel filtration of the purified enzyme yielded a 83000 Da protein. Subunits of about 42000 Da were estimated from sodium dodecyl sulfate-polyacrylamide gels. Wheat leaf PRK was stable for at least four weeks when stored at 4°C. Saturation curves for ribulose 5-phosphate (Ru5P) and ATP followed Michaelis-Menten kinetics (K _m values: K _{m Ru5P}=50–80 M; K _{m ATP}=70 M). The saturation curve for MgCl₂ was sigmoidal (half-maximal velocity <0.5 mM). The affinity for Ru5P, ATP and Mg²⁺ was not affected by pH changes comparable to pH shifts in the stroma. In contrast to chloroplast fructose-bisphosphatase (Zimmermann et al. 1976, Eur. J. Biochem. 70, 361–367) the affinity for ligands remained unchanged in the dithiothreitol-activated and in the non-activated state. The activity of PRK was increasingly sensitive to inhibition by 3-phosphoglyceric acid with decreasing pH below pH 8.0.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - PRK phosphoribulokinase - Ru5P ribulose-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis     

Rapid alterations in growth rate and electrical potentials upon stem excision in pea seedlings

Excision of the epicotyl base of pea (Pisum sativum L.) seedlings in air results in a fast drop in the growth rate and rapid transient membrane depolarization of the surface cells near the cut. Subsequent immersion of the cut end into solution leads to a rapid, transient rise in the epicotyl growth rate and an acropetally propagating depolarization with an amplitude of about 35 mV and a speed of approx. 1 mm · s^–1. The same result can be achieved directly by excision of the pea epicotyl under water. Shape, amplitude and velocity of the depolarization characterize it as a slow-wave potential. These results indicate that the propagating depolarization is caused by a surge in water uptake. Neither a second surge in water uptake (measured as a rapid increase in growth rate when the cut end was placed in air and then back into solution) nor another cut can produce the depolarization a second time. Cyanide suppresses the electrical signal at the treated position without inhibiting its transmission through this area and its development in untreated parts of the epicotyl. The large depolarization and repolarization which occur in the epidermal and subepidermal cells are not associated with changes in cell input resistance. Both results indicate that it is a transient shut-down of the plasma-membrane proton pump rather than large ion fluxes which is causing the depolarization. We conclude that the slow wave potential is spread in the stem via a hydraulic surge occurring upon relief of the negative xylem pressure after the hydraulic resistance of the root has been removed by excision.Abbreviations and Symbols GR growth rate - P_x xylem pressure - R_in cell input resistance - SWP slow wave potential - V_m membrane potential - V_s surface potential This work was supported by grants to D.J.C. from the National Science Foundation and the U.S. Department of Energy.     

The photosynthetic induction response in wheat leaves: net CO2 uptake,enzyme activation,and leaf metabolites

The photosynthetic induction response was studied in whole leaves of wheat (Triticum aestivum L.) following 5-min, 30-min and 10-h dark periods. After the 5-min dark treatment there was a rapid burst in the rate of photosynthesis upon illumination (half of maximum after 30s), followed by a slight decrease after 1.5 more min and then a gradual rise to the maximum rate. During this initial burst in photosynthesis, there was a rapid rise in the level of 3-phosphoglycerate (PGA) and a high PGA/triose-phosphate (triose-P) ratio was obtained. In addition, after the 5-min dark treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39), ribulose-5-phosphate kinase (EC 2.7.1.19) and chloroplastic fructose-1,6-bisphosphatase (EC 3.1.3.11) maintained a relatively high state of activation, and maximum activation occurred within 1 min of illumination. The results indicate there is a high capacity for CO₂ fixation in the cycle upon illumination but attaining maximum rates requires an increase in the ribulose-1,5-bisphosphate (RuBP) pool (adjustment in triose-P utilization for carbohydrate synthesis versus RuBP synthesis). With both the 30-min and 10-h dark pretreatments there was only a slight rise in photosynthesis upon illumination, followed by a lag, then a gradual increase to steady-state (half-maximum rate after 6 min). In contrast to the 5-min dark treatment, the level of PGA was low and actually decreased initially, whereas the level of RuBP increased and was high during induction, indicating that Rubisco is limiting. This regulation via the carboxylase was not reflected in the initial extractable activity, which reached a maximum by 1 min after illumination. The light activation of chloroplastic fructose-1,6-bisphosphatase in leaves darkened for 30 min and 10 h prior to illumination was relatively slow (reaching a maximum after 8 min). However, this was not considered to limit carbon flux through the carbon-fixation cycle during induction since RuBP was not limiting. When photosynthesis approached the maximum steady-state rate, a high PGA/triose-P ratio and a high PGA/RuBP ratio were obtained. This may allow a high rate of photosynthesis by producing a favorable mass-action ratio for the reductive phase (the conversion of PGA to triose phosphate) while stimulating starch and sucrose synthesis.Abbreviations Chl chlorophyll - FBP fructose-1,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - Pi inoganic phosphate - Rubisco RuBP carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate - triose-P triose phosphates (dihydroxyacetone phosphate+glyceraldehyde-3-phosphate)     

Osmotic potential and abscisic acid regulate triacylglycerol synthesis in developing wheat embryos

This work was carried out to determine what factors in the developing wheat (Triticum aestivum L.) grain are involved in regulating the metabolism of the triacylglycerol (TAG) storage reserves. When embryos are isolated from the grain and incubated in media for 4 d the TAG content is affected in three ways. In the basal medium (dilute buffer) the content falls; in 30 mM sucrose the content remains unchanged; in sucrose supplemented with an osmoticum (400 mM mannitol) or abscisic acid (1 M ABA) the TAG content increases. Effective osmotic potentials and ABA concentrations fit well with their respective values in planta. The fatty-acid composition of TAG accumulated in vitro is close to that in planta but in the absence of ABA or osmoticum there is a fall in the C₁₈C₁₆ ratio. Experiments with [¹⁴C] acetate show that the in-planta rate of incorporation into TAG can only occur in isolated embryos treated osmotically or with ABA, while there seems to be no effect of these two factors on TAG breakdown. An osmotic shock (dilute buffer) for only 2 h causes a rapid fall in TAG synthesis which continues for ca. 24 h after which it recovers. Abscisic acid protects against osmotic shock. It is concluded that TAG synthesis in developing wheat embryos is regulated by the osmotic potential and-or ABA, and that the embryos are very sensitive to short-term perturbations of these two factors.Abbreviations ABA abscisic acid - dpa days post anthesis - TAG triacylglycerol We are grateful to the European Economic Community for a Fellowship to R.R.S. which provided financial support for this work.     

Lectin synthesis in developing and germinating wheat and rye embryos

Wheat (Triticum aestivum L.) and rye (Secale cereale L.) lectins are specifically synthesized during seed formation. They accumulate exponentially in the primary axes in a period coinciding with the development of this complex organ. Since the specific lectin content also increases dramatically, there is apparently an outburst of lectin synthesis during the development of the primary axes. Germinating embryos also synthesize some lectin. The fortunate availability of a highly specific procedure for the isolation of cereal lectins enabled us to follow the kinetics of their synthesis during early germination. Stored mRNAs appear to be involved in this residual lectin synthesis.     

The relationship between wound-induced proteinase inhibitors and hydraulic signals in tomato seedlings

Localized wounding is known to induce systemic proteinase inhibitors (PI) in seedlings of tomato (Lycopersicon esculentum L.). Inhibitors of elastase (EC 3.4.21.36) were shown here to be among those systemically induced by wounding, and a simple rapid assay for PI based on elastase was developed. Using this assay, the nature of the systemic signalling system (‘PIIF’) was investigated. Hydraulic signals were shown to be induced in tomato by localized wounds. These signals travelled throughout the plant well within the lag time before appearance of systemic wound-induced PI. A number of correlations were drawn between the occurrence of the hydraulic signals and induction of systemic PI, suggesting that hydraulic signals might be the PIIF, or a component of it. It was shown that systemic hydraulic signals could be triggered, without significant wounding, by excision of a single leaflet through the submerged petiole. These hydraulic signals were similar in both kinetics and magnitude to those induced by localized wounding. However, they did not induce systemic PI. In addition, it was shown that systemic events almost as rapid as wound-induced hydraulic signals could be induced without wounding, under certain environmental conditions. This indicates that rapid hydraulic signals do not provide a specific signal of wounding. These findings demonstrate that hydraulic signals per se are not the PIIF.     

Genetic control of triosephosphate isomerase isoenzymes in wheat and rye

Summary The patterns of chloroplastic and cytosolic isoenzymes of triosephosphate isomerase were analysed by immunoblotting in leaves of rye, wheat, and some species of Aegilops or Agropyrum. While rye contained solely one chloroplastic and one cytosolic isoenzyme, wheat had a much more complex pattern which can be explained by the presence of three genomes in 6 x wheats (AABBDD) with distinct triosephosphate isomerase genes that provided different subunit species for the dimeric isoenzyme molecules. The 6 × wheats contained five, the 4 × wheats three, and the 2 × wheats only one chloroplastic isoenzyme band. The isoenzyme patterns were in accordance with a potential origin of one of the three chloroplastic triosephosphate isomerase genes of 6 × wheats from an Aegilops ancestor. The descent of the other two genes was, however, not in accordance with common contentions on the general evolution of cultural wheats. In the reciprocal intergeneric hybrids Secalotricum and Triticale both the chloroplastic and the cytosolic isoenzyme patterns of rye and wheat were biparentally inherited, indicating that both isoenzymes were controlled by nuclear genes. When monitored by immunoblotting the chloroplastic triosephosphate isomerase isoenzymes may provide useful genetic markers.     

Influence of acetylcholine agonists and antagonists on the swelling of etiolated wheat (Triticum aestivum L.) mesophyll protoplasts

Etiolated wheat (Triticum aestivum L.) mesophyll protoplasts swell within 30 min in darkness after a red light (R) pulse or addition of acetylcholine (ACh), if 0.5 mM CaCl₂ is present in the medium. In addition, ACh is also able to induce swelling in the presence of both 0.1 mM KCl or NaCl. Besides ACh, only carbamylcholine out of the choline derivatives tested was active in induction of swelling in the presence of K⁺ or Na⁺. The K⁺/Na⁺-dependent ACh-induced protoplast swelling was nullified by a ‘calmodulin inhibitor’, but not by Ca²⁺-channel blockers, Li⁺ or VO ₄ ^3- . The antagonists atropine (of muscarine-sensitive ACh receptors, mAChRs) andd-tubocurarine (of nicotine-sensitive ACh receptors, nAChRs) nullified the Ca²⁺ — and the K⁺/Na⁺-dependent protoplast swelling responses, respectively, while having no effect on the Ca²⁺-dependent R-induced swelling response. Moreover, muscarine and nicotine mimicked ACh in the Ca²⁺- and K⁺/Na⁺-dependent swelling responses respectively. Just as is the case in animal cells, the proposed mAChRs appear to be associated with a phosphatidylinositol-dependent pathway, whereas the proposed nAChRs are phosphatidylinositol independent. Similarity between the action of ACh via the proposed mChRs and R via phytochrome in protoplast swelling indicates they share in common signal-transduction pathway. We dedicate this paper to Hans Mohr on the occasion of his 60th birthday We thank the Department of Molecular Biology of the Agricultural University, Wageningen for the use of the photomicroscope and Dr. G. Fassina, Department of Pharmacology, University of Padua, Italy for the gift of nifedipine. These studies were supported by The Foundation for Fundamental Biological Research (BION) which is subsidized by The Netherlands Organization for the Advancement of Research (NWO). A.T. was also supported by: a Research Fellowship from the Agricultural University, Wageningen; a Visitors Fellowship from NWO, the Netherlands; RP II 12.15 from Ministry of Education, Poland.     

Metabolism of R,S-[2-14C]abscisic acid by non-stressed and water-stressed detached leaves of wheat (Triticum aestivum L.)

Metabolism of R,S-[2-¹⁴C]abscisic acid (ABA) was studied in detached leaves of six wheat (Triticum aestivum) cultivars, using non-stressed leaves or leaves water stressed by desiccation to 90% of their original fresh weight. The rate constant of ABA metabolism was similar in nonstressed leaves of all cultivars. Water stress resulted in significantly lower rate constants in two cultivars which accumulated high levels of ABA when stressed, the constants decreasing by a factor of about 1.5. Rate constants for the remainder of the cultivars were not significantly different from those for the non-stressed controls. It was calculated that if decreased metabolism was the mechanism for the accumulation of ABA following water stress the rate constants of metabolism would have to be reduced by a factor of between 25 and 70. The results therefore support the hypothesis that enhanced synthesis rather than reduced degradation is the main process by which ABA levels are elevated following experimentally induced water stress. There were differences between the six cultivars in the products of ABA metabolism. Over the time period studied, oxidation to phaseic acid and dihydrophaseic acid as well as to other unidentified metabolites appeared to be the predominant pathway of ABA metabolism, rather than conjugation to ABA glucose ester and other more polar compounds.Abbreviations ABA abscisic acid - ABAGE abscisic-acid glucose ester - DPA dihydrophaseic acid - PA phaseic acid     

Induction and ionic basis of slow wave potentials in seedlings of Pisum sativum L.

Slow wave potentials (SWPs) are transient depolarizations which propagate substantial distances from their point of origin. They were induced in the epidermal cells of pea epicotyls by injurious methods such as root excision and heat treatment, as well as by externally applied defined steps in xylem pressure (Px) in the absence of wounding. The common principle of induction was a rapid increase in Px. Such a stimulus appeared under natural conditions after (i) bending of the epicotyl, (ii) wounding of the epidermis, (iii) rewatering of dehydrated roots, and (iv) embolism. The induced depolarization was not associated with a change in cell input resistance. This result and the ineffectiveness of ion channel blockers point to H(+)-pumps rather than ion channels as the ionic basis of the SWP. Stimuli such as excision, heat treatment and pressure steps, which generate SWPs, caused a transient increase in the fluorescence intensity of epicotyls loaded with the pH-indicator DM-NERF, a 2',7'-dimethyl derivative of rhodol, but not of those loaded with the pH indicator 2',7'bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Matching kinetics of depolarization and pH response identify a transient inactivation of proton pumps in the plasma membrane as the causal mechanism of the SWP. Feeding pump inhibitors to the cut surface of excised epicotyls failed to chemically simulate a SWP; cyanide, azide and 2,4-dinitrophenol caused sustained, local depolarizations which did not propagate. Of all tested substances, only sodium cholate caused a transient and propagating depolarization whose arrival in the growing region of the epicotyl coincided with a transient growth rate reduction.     

Gibberellins and leaf expansion in near-isogenic wheat lines containing Rht1 and Rht3 dwarfing alleles

In near-isogenic lines of winter wheat (Triticum aestivum L. cv. Maris Huntsman) grown at 20° C under long days the reduced-height genes, Rht1 (semi-dwarf) and Rht3 (dwarf) reduced the rate of extension of leaf 2 by 12% and 52%, respectively, compared with corresponding rht (tall) lines. Lowering the growing temperature from 20° to 10° C reduced the rate of linear extension of leaf 2 by 2.5-fold (60% reduction) in the rht3 line but by only 1.6-fold (36% reduction) in the Rht3 line. For both genotypes, the duration of leaf expansion was greater at the lower temperature so that final leaf length was reduced by only 35% in the rht3 line and was similar in the Rht3 line at both temperatures. Seedlings of the rht3 (tall) line growing at 20° C responded positively to root-applied gibberellin A₁ (GA₁) in the range 1–10 μM GA₁; there was a linear increase in sheath length of leaf 1 whereas the Rht3 (dwarf) line remained unresponsive. Gibberellins A_{1, 3, 4, 8, 19, 20, 29, 34, 44} and ₅₃ were identified by full-scan gas chromatography-mass spectrometry in aseptically grown 4-d-old shoots of the Rht3 line. In 12-d-old seedlings grown at 20° C, there were fourfold and 24-fold increases in the concentration of GA₁ in the leaf expansion zone of Rht1 and Rht3 lines, respectively, compared with corresponding rht lines. Although GA₃ was present at a similar level to GA₁ in the rht3 (tall) line it accumulated only fivefold in the Rht3 (dwarf) line. The steady-state pool sizes of endogenous GAs were GA₁₉ ? GA₂₀ = GA₁ in the GA-responsive rht3 line whereas in the GA non-responsive Rht3 line the content of GA₁₉≈ GA₂₀ ? GA₁. It is proposed that one of the consequences of GA₁ action is suppression of GA₁₉-oxidase activity such that the conversion of GA₁₉ to GA₂₀ becomes a rate-limiting step on the pathway to GA₁ in GA-responsive lines. In the GA-non-responsive Rht lines it is suggested that GA₁₉ oxidase is not downregulated to the same extent and GA₁ accumulates before the next rate-limiting step on the pathway, its 2β-hydroxylation to GA₈. The steady-state pool sizes of GA_{19, 20, 1, 3} and ₈ were similar in developmentally equivalent tissues of the rht3 (tall) line growing at 10° C and 20° C despite a 2.5-fold difference in the rate of leaf expansion. In contrast, in the Rht3 (dwarf) line, the extent of accumulation of GA₁ reflected the severity of the phenotype at the two temperatures with slower growing tissues accumulating less, not more, GA₁. These results are interpreted as supporting the proposed model of regulation of the GA-biosynthetic pathway rather than previous suggestions that GA₁ accumulates in GA-insensitive dwarfs as a consequence of reduced growth rates.