Showing papers in "Planta in 1992"

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II.

Abstract: The obligate shade plant, Tradescantia albiflora Kunth grown at 50 μmol photons · m(-2) s(-1) and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 μmol · m(-2) · s(-1), were exposed to photoinhibitory light conditions of 1700 μmol · m(-2) · s(-1) for 4 h at 22° C. Photosynthesis was assayed by measurement of CO2-saturated O2 evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 μmol · m(-2) · s(-1) was most resistant, with pea grown at 50 μmol · m(-2) · s(-1) showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (qp) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F'vF'm, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81-92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F'vF'm, since qp at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid ΔpH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to qp, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.

Growth of cotton under continuous salinity stress: influence on allocation pattern, stomatal and non-stomatal components of photosynthesis and dissipation of excess light energy.

Abstract: Cotton (Gossypium hirsutum L.) plants were grown in flowing-culture solutions containing 0%, 26% and 55% natural seawater under controlled and otherwise identical conditions. Leaf Na(+) content rose to 360 mM in 55% seawater, yet the K(+) content was maintained above 100 mM. The K(+)/Na(+) selectivity ratio was much greater in the saline plants than in the control plants. All plants were healthy and able to complete the life cycle but relative growth rate fell by 46% in 26% seawater and by 83% in 55% seawater. Much of this reduction in growth was caused by a decreased allocation of carbon to leaf growth versus root growth. The ratio of leaf area/plant dry weight fell by 32% in 26% seawater and by 50% in 55 % seawater while the rate of carbon gain per unit leaf area fell by only 20% in 26% seawater and by as much as 66% in 55% seawater. Partial stomatal closure accounted for nearly all of the fall in the photosynthesis rate in 26% seawater but in 55% seawater much of the fall also can be attributed to non-stomatal factors. As a result of the greater effect of salinity on stomatal conductance than on CO2-uptake rate, photosynthetic water-use efficiency was markedly improved by salinity. This was also confirmed by stablecarbon-isotope analyses of leaf sugar and of leaf cellulose and starch. - Although non-stomatal photosynthetic capacity at the growth light was reduced by as much as 42% in 55% seawater, no effects were detected on the intrinsic photon yield of photosynthesis nor on the efficiency of photosystem II photochemistry, chlorophyll a/b ratio, carotenoid composition or the operation of the xanthophyll cycle. Whereas salinity caused in increase in mesophyll thickness and content of chloroplast pigments it caused a decrease in total leaf nitrogen content. The results indicate that the salinity-induced reduction in non-stomatal photosynthetic capacity was not caused by any detrimental effect on the photosynthetic apparatus but reflects a decreased allocation to enzymes of carbon fixation. - Rates of energy dissipation via CO2 fixation and photorespiration, calculated from gas-exchange measurements, were insufficient to balance the rate of light-energy absorption at the growth light. Salinity therefore would have been expected to cause the excess excitation energy to rise, leading to an increased nonradiative dissipation in the pigment bed and resulting increases in non-photochemical fluorescence quenching and zeaxanthin formation. However, no such changes could be detected, implying that salinity may have increased energy dissipation via a yet unidentified energy-consuming process. This lack of a response to salinity stress is in contrast to the responses elicited by short-term water stress which caused strong non-photochemical quenching and massive zeaxanthin formation.

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with ‘antisense’ rbcS

Abstract: The effect of nitrogen supply during growth on the contribution of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; EC 4.1.1.39) to the control of photosynthesis was examined in tobacco (Nicotiana tabacum L.). Transgenic plants transformed with antisense rbcS to produce a series of plants with a progressive decrease in the amount of Rubisco were used to allow the calculation of the flux-control coefficient of Rubisco for photosynthesis (CR). Several points emerged from the data: (i) The strength of Rubisco control of photosynthesis, as measured by CR, was altered by changes in the short-term environmental conditions. Generally, CR was increased in conditions of increased irradiance or decreased CO2. (ii) The amount of Rubisco in wild-type plants was reduced as the nitrogen supply during growth was reduced and this was associated with an increase in CR. This implied that there was a specific reduction in the amount of Rubisco compared with other components of the photosynthetic machinery. (iii) Plants grown with low nitrogen and which had genetically reduced levels of Rubisco had a higher chlorophyll content and a lower chlorophyll a/b ratio than wild-type plants. This indicated that the nitrogen made available by genetically reducing the amount of Rubisco had been re-allocated to other cellular components including light-harvesting and electron-transport proteins. It is argued that there is a “luxury” additional investment of nitrogen into Rubisco in tobacco plants grown in high nitrogen, and that Rubisco can also be considered a nitrogen-store, all be it one where the opportunity cost of the nitrogen storage is higher than in a non-functional storage protein (i.e. it allows for a slightly higher water-use efficiency and for photosynthesis to respond to temporarily high irradiance).

Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca(2+) and protein-kinase activity.

Abstract: Cell-wall components from the ectomycorrhizal fungi Amanita muscaria and Hebeloma crustuliniforme and from the spruce pathogen Heterobasidion annosum elicited a transient release of active oxygen species from cultured spruce cells (Picea abies (L.) Karst.). Since the detection of active oxygen was suppressed by catalase, H2O2 was assumed to be the prevailing O2 species. On the other hand, superoxide dismutase enhanced the concentration of detectable H2O2 indicating that the superoxide anion was formed before dismutating to H2O2. The elicitors induced the formation of active oxygen in a dose-dependent manner. Interestingly, elicitors from mycorrhizal fungi had a lower H2O2-inducing activity than equal amounts of cell-wall preparations from the pathogen H. annosum. In Ca2+-depleted medium the production of active oxygen by elicitor-treated spruce cells was suppressed. Additionally, the ionophore A 23187 induced active oxygen formation in a medium with Ca2+ but not in a Ca2+-depleted medium. Furthermore, the protein-kinase inhibitor staurosporine inhibited the oxidative burst. At a concentration of 34 nM the effect was diminished to 50%. From these results it is suggested that the release of active oxygen species from cultured spruce cells triggered by cell-wall-derived fungal elicitors depends on external Ca2+ and a protein-kinase activity. In these respects the effect shows similarities with the well-studied respiratory burst of mammalian neutrophils.

Regeneration in Phaseolus vulgaris L.: High-frequency induction of direct shoot formation in intact seedlings by N(6)-benzylaminopurine and thidiazuron.

Abstract: Induction of prolific shoot formation in Phaseolus vulgaris L. cv. Kinghorn Wax was achieved by germinating mature seeds and growing seedlings on a medium supplemented with 10 μM thidiazuron (TDZ), a substituted phenylurea, or 80 μM N6-benzylaminopurine (BAP). Culture for 7 d in the presence of 10 μM TDZ was sufficient to induce maximal shoot formation, whereas a continuous presence of BAP was required for the induction and development of shoots. The differentiation of adventitious shoots occurred within four weeks of seed culture, from tissues in the regions of axillary buds on the cotyledonary node and also areas surrounding the shoot apex of the intact seedling. The number of shoots regenerated from intact seedlings was significantly higher than that obtained with expiants. Regenerated shoots developed into flowering plants. Similar results were obtained in six other bean cultivars.

Operation of the xanthophyll cycle in higher plants in response to diurnal changes in incident sunlight

Abstract: Changes in the carotenoid composition of leaves in response to diurnal changes in sunlight were determined in the crop species Helianthus annuus L. (sunflower), Cucurbita pepo L. (pumpkin), and Cucumls sativus L. (cucumber), in the diaheliotropic mesophyte Malva neglecta Wallr., and in the perennial shrub Euonymus kiautschovicus Loesner. Large daily changes were observed in the relative proportions of the components of the xanthophyll cycle, violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) in plants grown in full sunlight. In all leaves large amounts of Z were formed at peak irradiance, with the changes in Z content closely following changes in incident photon flux density (PFD) over the course of the day. All leaves also contained large total pools of the three xanthophyll-cycle components. However, the extent to which the V pool present at dawn became de-epoxidized during the day varied widely among leaves, from a 27% decrease in M. neglecta to a 90% decrease in E. kiautschovicus. The largest amounts of Z and the lowest amounts of V at peak irradiance (full sunlight) were observed in the species with the lower rates of photosynthesis (particularly in E. kiautschovicus and pumpkin), and smaller amounts of Z and a lesser decrease in V content were found at peak irradiance in those species with the higher rates of photosynthesis (particularly in M. neglecta and sunflower). In all species some Z was present in the leaves prior to sunrise. Furthermore, in individuals of sunflower, pumpkin, and cucumber grown at 85% of full sunlight and transferred to full sunlight, a further increase in the already large pool of the xanthophyll-cycle pigments occurred over the course of 1 d.

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Abstract: Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.

Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

Abstract: The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.

Effect of anoxia on starch breakdown in rice and wheat seeds

Abstract: The capabilities of rice (Oryza sativa L.) and wheat (Triticum aestivum L.) seeds (caryopses) to degrade starchy reserves present in the endosperm tissue were compared under anaerobic conditions. The results showed that rice, a species highly tolerant to anoxia, can readily break down starch under anaerobiosis concomitant with germination, while wheat does not germinate and fails to degrade starch present in the endosperm. This clearly distinct behavior is likely the consequence of the successful inducible formation of α-amylase (EC 3.2.1.1.) in rice under anoxia, whereas the enzyme is not produced in wheat seeds. We found that rice seeds possess a set of enzymes allowing starch and its degradative products to be utilized under anoxic conditions. Wheat seeds were shown to germinate even under anoxia if fed glucose or sucrose exogenously. The overall results indicate that induction of α-amylase appears to be one of the factors permiting rice seeds to germinate in totally anaerobic environments.

Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa.

Abstract: Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional relations of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5-2 μm) sections of material embedded in butyl-methylmethacrylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana(L.) Heynh, Pinus radiataD. Don, Zamia furfuraceaAit., Azolla pinnataR. Br. and on sporophytic tissues of Funaria hygrometricaHedw. In general, most of the cells in the organs studied are successfully stained. Using this method, we find that interphase meristematic cells in all of these species have microtubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the calcium chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid EGTA in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant microtubules during fixation appears unwarranted.

Mechanistic differences in photoinhibition of sun and shade plants.

Abstract: Leaf discs of the shade plant Tradescantia albiflora Kunth grown at 50 mumol . m-2 . s-1, and the facultative sun, shade plant Pisum sativum L. grown at 50 or 300 mumol . m-2 . s-1, were photoinhib ...

Two components of onset and recovery during photoinhibition of Ulva rotundata.

Abstract: Short-term (up to 5 h) transfers of shade-adapted (100 μmol · m−2 · s−1) clonal tissue of the marine macroalga Ulva rotundata Blid. (Chlorophyta) to higher irradiances (1700, 850, and 350 μmol · m−2 · s−1) led to photoinhibition of room-temperature chlorophyll fluorescence and O2 evolution. The ratio of variable to maximum (Fv/Fm) and variable (Fv) fluorescence, and quantum yield (ϕ) declined with increasing irradiance and duration of exposure. This decline could be resolved into two components, consistent with the separation of photoinhibition into energy-dissipative processes (photoprotection) and damage to photosystem II (PSII) by excess excitation. The first component, a rapid decrease in Fv/Fm and in Fv, corresponds to an increase in initial (Fo) fluorescence and is highly sensitive to 1 mM chloramphenicol. This component is rapidly reversible under dim (40 μmol · m−2 · s−1) light, but is less reversible with increasing duration of exposure, and may reflect damage to PSII. The second (after 1 h exposure) component, a slower decline in Fv/Fm and Fv with declining Fo, appears to be associated with the photoprotective interconversion of violaxanthin to zeaxanthin and is sensitive to dithiothreitol. The accumulation of zeaxanthin in U. rotundata is very slow, and may account for the predominance of increases in Fo at high irradiances.

Morphology and Monoterpene Biosynthetic Capabilities of Secretory Cell Clusters Isolated from Glandular Trichomes of Peppermint (Mentha Piperita L)

Abstract: Secretory cells were isolated from the monoterpene-producing glandular trichomes (peltate form) of peppermint as clusters of eight cells each. These isolated structures were shown to be non-specifically permeable to low-molecular-weight, water-soluble cofactors and substrates. Short incubation periods with the polar dye Lucifer yellow iodoacetamide (Mr=660) resulted in a uniform staining of the cytoplasm, with exclusion of the dye from the vacuole. The molecular-weight exclusion limit for this permeability was shown to be less than approx. 1800, based on exclusion of fluorescein-conjugated dextran (Mr ∼ 1800). Intact secretory cell clusters very efficiently incorporated [3H]geranyl pyrophosphate into monoterpenes. The addition of exogenous cofactors and redox substrates affected the distribution of monoterpenes synthesized from [3H]geranyl pyrophosphate, demonstrating that the cell clusters were permeable to these compounds and that the levels of endogenous cofactors and redox substrates were depleted in the isolated cells. When provided with the appropriate cofactors, such as NADPH, NAD+, ATP, ADP and coenzyme A, the isolated secretory cell clusters incorporated [14C]sucrose into monoterpenes, indicating that these structures are capable of the de-novo biosynthesis of monoterpenes from a primary carbon source, and that they maintain a high degree of metabolic competence in spite of their permeable nature.

Fungal elicitor-mediated responses in pine cell cultures I. Induction of phenylpropanoid metabolism

Abstract: A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin βglucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.

Characterization of the plasma-membrane H(+)-ATPase from Vicia faba guard cells : Modulation by extracellular factors and seasonal changes.

Abstract: Stomatal movement is controlled by external and internal signals such as light, phytohormones or cytoplasmic Ca(2+). Using Vicia faba L., we have studied the dose-dependent effect of auxins on the modulation of stomatal opening, mediated through the activity of the plasma-membrane H(+)-ATPase. The patch-clamp technique was used to elucidate the electrical properties of the H(+)-ATPase as effected by growth regulators and seasonal changes. The solute composition of cytoplasmic and extracellular media was selected to record pump currents directly with high resolution. Proton currents through the ATPase were characterized by a voltage-dependent increase in amplitude, positive to the resting potential, reaching a plateau at more depolarized values. Upon changes in extracellular pH, the resting potential of the cell shifted with a non-Nernst potential response (±21 mV), indicating the contribution of a depolarizing ionic conductance other than protons to the permeability of the plasma membrane. The use of selective inhibitors enabled us to identify the currents superimposing the H(+)-pump as carried by Ca(2+). Auxinstimulation of this electroenzyme resulted in a rise in the outwardly directed H(+) current and membrane hyperpolarization, indicating that modulation of the ATPase by the hormone may precede salt accumulation as well as volume and turgor increase. Annual cycles in pump activity (1.5-3.8 μA · cm(-2)) were expressed by a minimum in pump current during January and February. Resting potentials of up to -260 mV and plasmamembrane surface area, on the other hand, did not exhibit seasonal changes. The pump activity per unit surface area was approximately 2- to 3-fold higher in guard cells than in mesophyll cells and thus correlates with their physiological demands.

Calcium influx at the tip of growing root-hair cells of Arabidopsis thaliana.

Abstract: The role of extracellular Ca2+ in root-hair tip growth has been investigated in Arabidopsis thaliana (L.) Heynh. Root-hair length was found to be dependent on the concentration of Ca2+ in the growth medium, with maximum length achieved at [Ca2+] of 0.3–3.0 mM. Using a non-intrusive calcium-specific vibrating microelectrode, an extracellular Ca2+ gradient was detected at the tips of individual growing root-hair cells. The direction of the gradient indicated a net influx of Ca2+ into root-hair cells. No gradient was detected near the sides of the root hairs or at the tips of non-growing root hairs. When root hairs were exposed to the Ca2+-channel blocker nifedipine, tip growth stopped and the extracellular Ca2+ gradient was abolished. These results indicate that Ca2+ influx through plasma-membrane Ca2+ channels is required for normal root-hair tip growth.

Asparagine metabolism and nitrogen distribution during protein degradation in sugar-starved maize root tips.

Abstract: Excised maize (Zea mays L.) root tips were used to monitor the effects of prolonged glucose starvation on nitrogen metabolism. Following root-tip excision, sugar content was rapidly exhausted, and protein content declined to 40 and 8% of its initial value after 96 and 192 h, respectively. During starvation the contents of free amino acids changed. Amino acids that belonged to the same “synthetic family” showed a similar pattern of changes, indicating that their content, during starvation, is controlled mainly at the level of their common biosynthetic steps. Asparagine, which is a good marker of protein and amino-acid degradation under stress conditions, accumulated considerably until 45 h of starvation and accounted for 50% of the nitrogen released by protein degradation at that time. After 45 h of starvation, nitrogen ceased to be stored in asparagine and was excreted from the cell, first as ammonia until 90–100 h and then, when starvation had become irreversible, as amino acids and aminated compounds. The study of asparagine metabolism and nitrogen-assimilation pathways throughout starvation showed that: (i) asparagine synthesis occurred via asparagine synthetase (EC 6.3.1.1) rather than asparagine aminotransferase (EC 2.6.1.14) or the β-cyanoalanine pathway, and asparagine degradation occurred via asparaginase (EC 3.5.1.1); and (ii) the enzymic activities related to nitrogen reduction and assimilation and amino-acid synthesis decreased continuously, whereas glutamate dehydrogenase (EC 1.4.1.2–4) activities increased during the reversible period of starvation. Considered together, metabolite analysis and enzymic-activity measurements showed that starvation may be divided into three phases: (i) the acclimation phase (0 to 30–35 h) in which the root tips adapt to transient sugar deprivation and partly store the nitrogen released by protein degradation, (ii) the survival phase (30–35 to 90–100 h) in which the root tips expel the nitrogen released by protein degradation and starvation may be reversed by sugar addition and (iii) the cell-disorganization phase (beyond 100 h) in which all metabolites and enzymic activities decrease and the root tips die.

Manipulation of conditions for the culture of somatic embryos of white spruce for improved triacylglycerol biosynthesis and desiccation tolerance.

Abstract: In order to enhance post-germinative vigour, somatic embryos of Picea glauca (Moench) Voss. were matured under in-vitro conditions that stimulated triacylglycerol (TAG) biosynthesis. In P. glauca seeds over 90% of the TAG was stored within the megagametophyte, and isolated zygotic embryos contained twice the amount of TAG of somatic embryos cultured for four weeks on basal medium containing 16 μM abscisic acid (ABA). Polyethylene glycol-4000 (PEG) as a non-permeating osmoticum with ABA promoted TAG biosynthesis by somatic embryos and sustained maturation throughout an eight-week culture period. Treatments that promoted TAG biosynthesis also prevented precocious germination and promoted desiccation tolerance. Thus, the optimal culture conditions for maturation, desiccation survival, and plantlet regeneration were 16–24 μM ABA and 7.5% PEG for eight weeks, followed by desiccation. Under these conditions the levels of TAG per somatic embryo were raised ninefold to about five times the zygotic-embryo level, and the TAG fatty-acid composition became similar to that of zygotic embryos. A study of sectioned material, using light and transmission electron microscopy, showed that the structure and distribution of lipid bodies within these somatic embryos and the degree of embryo development were similar to mature zygotic embryos. Up to 81% of the desiccated somatic embryos regenerated to plantlets during which time the TAG was utilised in a manner similar to zygotic seedlings.

A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L.

Abstract: A new method is described for the isolation of large quantities of Vicia faba metaphase chromo- somes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G1/S interface. After release from the block, the cells re-en- tered the cell cycle with a high degree of synchrony. A treatment with 2.5 p~M amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, exam- ined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30min. Under these conditions, 1.106 chromosomes were routinely obtained from 30 root tips. The chromo- somes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cyto- metric analysis and sorting. After the addition of hexy- lene glycol, the chromosome suspensions could be stored at 4 ~ C for six months without any signs of deterioration.

Hydration-state-responsive proteins link cold and drought stress in spinach.

Abstract: Spinach (Spinacia oleracea L) seedlings exposed to low nonfreezing temperatures (0–10° C) that promote cold acclimation, synthesize a variety cold-acclimation proteins and at the same time acquire a greater ability to withstand cellular dehydration imposed by the freezing of tissue water Two of these proteins (160 and 85 kDa) become more abundant over time at low temperature In addition, a small decline in tissue water status from a maximally hydrated state also appears to be associated with an initiation of the accumulation of these proteins at a noninductive temperature Imposing a severe water stress on young seedlings grown at 25° C by withholding water leads to substantial accumulation of the 160- and 85-kDa proteins, and maximal induction of freezing tolerance This evidence implies that responses to cold acclimation and water stress involve common mechanisms, and further establishes the linkage of these two proteins with stresses having an osmotic component

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex : Quantification of calcium fluxes using a calcium-selective vibrating microelectrode.

Abstract: The effects of aluminum on the concentration-dependent kinetics of Ca(2+) uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 μM CaCl2 solution (pH 4.5) for 3 d. Subsequently, net Ca(2+) fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca(2+)-selective microelectrode. The kinetics of Ca(2+) uptake into cells of the root apex, for external Ca(2+) concentrations from 20 to 300 μM, were found to be quite similar for both cultivars in the absence of external Al; Ca(2+) transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 μM total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca(2+) absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca(2+) uptake over the entire Ca(2+) concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca(2+) uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 μM) caused the K m for Ca(2+) uptake to increase with each rise in Al concentration, from approx. 100 μM in the absence of Al to approx. 300 μM in the presence of 10 μM Al, while having no effect on the V max. The same Al exposures had little effect on the kinetics of Ca(2+) uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca(2+) transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca(2+)-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.

Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide L-phosphinothricin (glufosinate) differently from untransformed plants.

Abstract: l-Phosphinothricin (l-Pt)-resistant plants were constructed by introducing a modified phosphinothricin-N-acetyl-transferase gene (pat) via Agrobacterium-mediated gene transfer into tobacco (Nicotiana tabacum L), and via direct gene transfer into carrot (Daucus carota L). The metabolism of l-Pt was studied in these transgenic, Pt-resistant plants, as well as in the untransformed species. The degradation of l-Pt, 14C-labeled specifically at different C-atoms, was analysed by measuring the release of 14CO2 and by separating the labeled degradation products on thin-layer-chromatography plates. In untransformed tobacco and carrot plants, l-Pt was deaminated to form its corresponding oxo acid 4-methylphosphinico-2-oxo-butanoic acid (PPO), which subsequently was decarboxylated to form 3-methylphosphinico-propanoic acid (MPP). This compound was stable in plants. A third metabolite remained unidentified. The l-Pt was rapidly N-acetylated in herbicide-resistant tobacco and carrot plants, indicating that the degradation pathway of l-Pt into PPO and MPP was blocked. The N-acetylated product, l-N-acetyl-Pt remained stable with regard to degradation, but was found to exist in a second modified form. In addition, there was a pH-dependent, reversible change in the mobility of l-N-acetyl-Pt thin-layer during chromatography.

Gibberellin-induced growth and regulation of the cell division cycle in deepwater rice.

Abstract: Excised stem sections of deepwater rice (Oryza sativa L., cv. Pin Gaew 56) containing the highest internode were used to study induction of rapid internodal elongation by gibberellin. It has been shown previously that this growth response is based on an increased cell production rate in the intercalary meristem and on increased cell elongation. Our investigations were aimed at establishing the temporal sequence of these GA-regulated processes. Cell sizes were determined by scanning electron microscopy, the phases of the cell cycle by flow cytometry, and DNA synthesis by [3H]thymidine incorporation. The lag time for the onset of gibberellic acid (GA3)-induced growth was 40 min. Treatment with GA3 promoted cell elongation in the intercalary meristem within 2 h. After 4 h of treatment with GA3, the fraction of meristematic cells in the G2 phase had declined, indicating that cells in the G2 phase had entered mitosis. Subsequent activation of DNA replication led to an overall increase in the cell-production rate. This was evident from an increase in the percentage of cells in the S phase and from enhanced incorporation of [3H]thymidine into DNA between 4 and 7 h of GA3 treatment. An increase in the final cell length contributed to the growth response after 7 h of GA3 application. Our results are consistent with the hypothesis that gibberellin first promotes cell elongation in the intercalary meristem and that cell division is stimulated as a result of cell-growth.

Biosynthesis of p-hydroxybenzoic acid in elicitor-treated carrot cell cultures.

Abstract: Carrot (Daucus carota L.) cells respond to treatment with fungal elicitors by synthesizing wallbound p-hydroxybenzoic acid (p-HBA). The biosynthetic pathway to p-HBA is still hypothetical. Tracer experiments with l-phenylalanine indicate the involvement of the general phenylpropanoid pathway. 3,4 (Methylenedioxy) innamic acid, an inhibitor of hydrocycinnamate CoA ligase, inhibits the accumulation of anthocyanins in carrot, while it does not interfere with p-HBA synthesis. Thus p-HBA biosynthesis does not appear to involve CoA thioesters. In the present report the sequence of enzymic reactions leading to p-HBA was investigated in vitro using protein preparations from cells treated with a fungal elicitor from Pythium aphanidermatum (Edson) Fitzp. The side-chain degradation from p-coumaric acid to p-HBA is not analogous to the β-oxidation of fatty acids and involves p-hydroxybenzaldehyde as an intermediate. The final step from p-hydroxybenzaldehyde to p-HBA is catalyzed by an NAD-dependent p-hydroxybenzaldehyde dehydrogenase (EC 1.2.1.-). This reaction was characterized with regard to cofactor requirements, pH and temperature optima. The in-vitro formation of p-HBA from p-coumaric acid and the activity of the hydroxybenzaldehyde dehydrogenase are moderately elicitor-induced but to a much lesser extent than phenylalanine ammonialyase, which is the starting enzyme of the general phenylpropanoid pathway.

Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem.

Abstract: Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions of Eucalyptus gunii Hook and partially characterized. They differ greatly in terms of both physical and biochemical properties, and can be separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The native molecular weight of of CAD 1 is 38 kDa as determined by gel-filtration chromatography on Superose 6, and this isoform is likely to be a monomer since it yields a polypeptide of 35 kDa upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It has a low substrate affinity for coniferyl and p-coumaryl alcohols and their corresponding aldehydes. No activity with sinapyl aldehyde and alcohol was detected. The more abundant isoform is CAD 2, which has a native molecular weight of 83 kDa and is a dinier composed of two subunits of slightly different molecular weights (42-43 kDa). These subunits show identical peptide patterns after digestion with N-chlorosuccinimide. The isoform, CAD 2, has a high substrate affinity for all the substrates tested. The two isoforms are immunologically distinct as polyclonal antibodies raised against CAD 2 do not cross-react with CAD 1. The characterization of two forms of CAD exhibiting such marked differences indicates their involvement in specific pathways of monolignol utilisation.

Distribution and accumulation of ultraviolet-radiation-absorbing compounds in leaves of tropical mangroves

Abstract: Ultraviolet (UV)-absorbing phenolic compounds that have been shown to be protective against the damaging: effects of UV-B radiation (Tevini et al., 1991, Photochem. Photobiol. 53, 329-333) were found in the leaf epidermis of tropical mangrove tree species. These UV-absorbing phenolic compounds and leaf succulence function as selective filters, removing short and energetic wavelengths. A field survey showed that the concentration of UV-absorbing compounds varied between species, between sites that would be experiencing similar levels of UV radiation, and between sun and shade leaves. Sun leaves have greater contents of phenolic compounds than shade leaves, and more saline sites have plants with greater levels in their leaves than less saline sites. In addition, increases in leaf nitrogen contents and quantum yields did not correlate with increasing levels of UV-absorbing compounds. It was concluded from these results that although UV-absorbing compounds form a UV-screen in the epidermis of mangrove leaves, UV radiation may not be the only factor influencing the accumulation of phenolic compounds, thus an experiment which altered the level of UV radiation incident on mangrove species was done. Near ambient levels of UVA and UV-B radiation resulted in a greater content of UV-absorbing compounds in Bruguiera parviflora (Roxb.) Wight and Arn. ex Griff., but did not result in increases in B. gymnorrhiza (L.) Lamk or Rhizophora apiculata Blume. Total chlorophyll contents were lower in R. apiculata when it was grown under near-ambient levels of UV radiation than when it was grown under conditions of UV-A and UV-B depletion, but no differences were observed between the UV radiation treatments in the other two species. There was no difference in leaf morphology, carotenoid/chlorophyll ratios, or chlorophyll a/b ratios between UV treatments, although these varied among species; B. parviflora had the highest carotenoid/chlorophyll ratio and R. apiculata had the lowest. Thus it is proposed that differences in species response tu UV radiation may be influenced by their ability to dissipate excess visible solar radiation.

Homoiohydrous (recalcitrant) seeds: Developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer.

Abstract: The desiccation sensitivity in relation to the stage of development was investigated in embryonic axes from the homoiohydrous (recalcitrant) seeds of Landolphia kirkii. Electrolyte leakage, used to assess membrane damage after flash (very rapid) drying, indicated that axes from immature (non-germinable) seeds were the most desiccation-tolerant, followed by those from mature seeds, while axes from seeds germinated for increasing times were progressively more desiccation-sensitive. Differential scanning calorimetry was used to study the relationship between desiccation sensitivity and the properties of water in the tissues. Axes from immature seeds had a lower content of non-freezable water than that of any other developmental stage and a higher enthalpy of melting of freezable water. For mature and immature axes electrolyte leakage increased at the point of loss of freezable water. At other developmental stages the water content at which electrolyte leakage increased markedly correlated with the other properties of the water, such as the change in the shape of the melting endotherm and the onset temperature. Ultrastructural studies of axes at the various developmental stages showed changes in the degree and pattern of vacuolation, the presence and quantities of lipid and starch, and the degree of endomembrane development. The results are discussed in relation to current hypotheses on the basis of desiccation tolerance.

Use of an extracellular, ion-selective, vibrating microelectrode system for the quantification of K(+), H (+), and Ca (2+) fluxes in maize roots and maize suspension cells.

Abstract: An ion-selective vibrating-microelectrode system, which was originally used to measure extracellular Ca(2+) gradients generated by Ca(2+) currents, was used to study K(+), H(+) and Ca(2+) transport in intact maize (Zea mays L.) roots and individual maize suspension cells. Comparisons were made between the vibrating ion-selective microelectrode, and a technique using stationary ion-selective microelectrodes to measure ionic gradients in the unstirred layer at the surface of plant roots. The vibrating-microelectrode system was shown to be a major improvement over stationary ion-selective microelectrodes, in terms of sensitivity and temporal resolution. With the vibrating ion microelectrode, it was easy to monitor K(+) influxes into maize roots in a background K(+) concentration of 10 mM or more, while stationary K(+) electrodes were limited to measurements in a background K(+) concentration of 0.3 mM or less. Also, with this system it was possible to conduct a detailed study of root Ca(2+) transport, which was previously not possible because of the small fluxes involved. For example, we were able to investigate the effect of the excision of maize roots on Ca(2+) influx. When an intact maize root was excised from the seedling at a position 3 cm from the site of measurement of Ca(2+) transport, a rapid fourfold stimulation of Ca(2+) influx was observed followed by dramatic oscillations in Ca(2+) flux, oscillating between Ca(2+) influx and efflux. These results clearly demonstrate that wound or perturbation responses of plant organs involve transient alterations in Ca(2+) transport, which had previously been inferred by demonstrations of touch-induced changes in cytoplasmic calcium. The sensitivity of this system allows for the measurement of ion fluxes in individual plant cells. Using vibrating K(+) and H(+)electrodes, it was possible to measure H(+)efflux and both K(+) influx and efflux in individual maize suspension cells under different conditions. The availability of this technique will greatly improve our ability to study ion transport at the cellular level, in intact plant tissues and organs, and in specialized cells, such as root hairs or guard cells.

Carbohydrate metabolism during postharvest ripening in kiwifruit.

Abstract: Mature fruit (kiwifruit) of Actinidia deliciosa var. deliciosa (A. Chev.), (C.F.) Liang and Ferguson cv. Haywood (Chinese gooseberry) were harvested and allowed to ripen in the dark at 20° C. Changes were recorded in metabolites, starch and sugars, adenine nucleotides, respiration, and sucrose and glycolytic enzymes during the initiation of starch degradation, net starch-to-sucrose conversion and the respiratory climacteric. The conversion of starch to sucrose was not accompanied by a consistent increase in hexose-phosphates, and UDP-glucose declined. The activity of sucrose phosphate synthase (SPS) measured with saturating substrate rose soon after harvesting and long before net sucrose synthesis commenced. The onset of sugar accumulation correlated with an increase in SPS activity measured with limiting substrates. Throughout ripening, until sucrose accumulation ceased, feeding [14C] glucose led to labelling of sucrose and fructose, providing evidence for a cycle of sucrose synthesis and degradation. It is suggested that activation of SPS, amplified by futile cycles, may regulate the conversion of starch to sugars. The respiratory climacteric was delayed, compared with net starchsugar interconversion, and was accompanied by a general decline of pyruvate and all the glycolytic intermediates except fructose-1,6-bisphosphate. The ATP/ ADP ratio was maintained or even increased. It is argued that the respiratory climacteric cannot be simply a consequence of increased availability of respiratory substrate during starch-sugar conversion, nor can it result from an increased demand for ATP during this process.

Aspartic proteinase from barley grains is related to mammalian lysosomal cathepsin D.

Abstract: Resting barley (Hordeum vulgare L.) grains contain acid-proteinase activity. The corresponding enzyme was purified from grain extracts by affinity chromatography on a pepstatin-Sepharose column. The pH optimum of the affinity-purified enzyme was between 3.5 and 3.9 as measured by hemoglobin hydrolysis and the enzymatic activity was completely inhibited by pepstatin a specific inhibitor of aspartic proteinases (EC 3.4.23). Further purification on a Mono S column followed by activity measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the affinity-purified enzyme preparation contained two active heterodimeric aspartic proteinases: a larger 48k Da enzyme, consisting of 32-kDa and 16-kDa subunits and a smaller one of 40 kDa, consisting of 29-kDa and 11-kDa subunits. Separation and partial amino acid sequence analysis of each subunit indicate that the 40-kDa enzyme is formed by proteolytic processing of the 48k Da form. Amino-acid sequence alignment and inhibition studies showed that the barley aspartic proteinase resembles mammalian lysosomal cathepsin D (EC 3.4.23.5).