Modulation by phytochrome of the blue light-induced extracellular acidification by leaf epidermal cells of pea (Pisum sativum l.): a kinetic analysis

Blue light induces extracellular acidification, a prerequisite of cell expansion, in epidermis cells of young pea leaves, by stimulation of the proton pumping-ATPase activity in the plasma membrane. A transient acidification, reaching a maximum 2.5-5 min after the start of the pulse, could be induced by pulses as short as 30 msec. A pulse of more than 3000 micromol m-2 saturated this response. Responsiveness to a second light pulse was recovered with a time constant of about 7 min. The fluence rate-dependent lag time and sigmoidal increase of the acidification suggested the involvement of several reactions between light perception and activation of the ATPase. In wild-type pea plants, the fluence response relation for short light pulses was biphasic, with a component that saturates at low fluence and one that saturates at high fluence. The phytochrome-deficient mutant pcd2 showed a selective loss of the high-fluence component, suggesting that the high-fluence component is phytochrome-dependent and the low-fluence component is phytochrome-independent. Treatment with the calmodulin inhibitor W7 also led to the elimination of the phytochrome-dependent high-fluence component. Simple models adapted from the one used to simulate blue light-induced guard cell opening failed to explain one or more elements of the experimental data. The hypothesis that phytochrome and a blue light receptor interact in a short-term photoresponse is endorsed by model calculations based upon a three-step signal transduction cascade, of which one component can be modulated by phytochrome.     

Calcium-calmodulin signalling is involved in light-induced acidification by epidermal leaf cells of pea, Pisum sativum L.

Pathways of signal transduction of red and blue light-dependentacidification by leaf epidermal cells were studied using epidermalstrips of the Argenteum mutant of Pisum sativum. In these preparationsthe contribution of guard cells to the acidification is minimal.The hydroxypyridine nifedipine, a Ca²⁺-channel blocker, partlyinhibited the response to both blue and red light, while thephenylalkylamine, verapamil, a Ca²⁺-channel blocker that hasbeen shown in plant cells also to block K⁺-channels, causednearly complete inhibition. The Ca²⁺-channel activator S(–)BayK 8644 induced acidification when added in the dark and diminishedthe light-induced lowering of the extracellular pH. The Ca²⁺-ionophores,ionomycin and A23187 [GenBank] , also reduced the light response. Furthermore,the light-induced acidification was inhibited by the calmodulinantagonists W-7 and trifluoperazine, but not by W-5. These calmodulininhibitors completely inhibited the red light-induced acidification,but inhibited the response to blue light by only 60–70%.In general, inhibition by compounds affecting Ca-calmodulinsignalling was always stronger on the red light response thanthat on the blue light response (with the exception of verapamilthat blocked both the red and blue light responses equally well).This differential effect on red and blue light-induced responsesindicates a role for Ca²⁺-CaM signalling in both the red andblue light responses, while a second process, independent ofCa²⁺ is activated by blue light. Key words: Signal transduction, light-induced acidification, epidermal cells, pea     

Light-dependent potassium uptake by Pisum sativum leaf fragments

A net K⁺ influx into chopped pea leaves bathed in 5 mM KCl,0.26 M sucrose and illuminated with 4000 lux amounted to about7.5 µmoles/g fresh weight-hr, while essentially no netflux occurred in the dark. This light-dependent K⁺ uptake waslinear with time for nearly 2 hr and continuously increasedas the light intensity was raised to 9000 lux. Over half ofthe K⁺ uptake was balanced by H⁺ release into the bathing solution,possibly by a mechanism in which bicarbonate was the anion accompanyingK⁺. The replacement of Cl^– by HCO₃^– increased thelight-dependent K⁺ uptake to 56 µmoles/g fresh weight-hr.About 23% of the light-dependent K⁺ uptake in 5 mM KCl was accompaniedby a Cl^– uptake. This net Cl^– influx was less sensitiveto the uncoupler tri-Fl-CCP and more sensitive to DCMU in thebathing solution than was the K⁺ uptake. The remaining net K⁺influx into pea leaf fragments was balanced by effluxes of sodium(accounting for 5%), magnesium (8%) and calcium (1%). (Received March 31, 1969; )     

Abscisic acid signal transduction in epidermal cells of Pisum sativum L. Argenteum: both dehydrin mRNA accumulation and stomatal responses require protein phosphorylation and dephosphorylation

The determinate growth of the primary root, its organization and relationship with lateral-root development, and the possible ecological significance of this growth pattern were analyzed in three sympatric species of Cactaceae from the Sonoran Desert, Stenocereus gummosus (Engelm.) Gibson & Horak, S. thurberi (Engelm.) Buxbaum and Ferocactus peninsulae (F.A.C. Weber) Britton & Rose, var. townsendianus (Britton & Rose) N.P. Taylor, stat. nov., Engelm. After seed germination, primary roots of these species commonly grew only for 2–3 d after the start of radicle protrusion (ASRP). This pattern of growth was observed on seedlings growing on filter paper, in vitro under sterile conditions, or in soil. The root-hair zone approached the very tip of the root and meristem exhaustion appeared to be typical in all seedlings of a population in all species. On average, 23 meristematic cells in the epidermal cell file in F. peninsulae were counted during the short steady-state period of growth (12–24 h ASRP). In S. gummosus, the size of the meristem was smaller with the number of epidermal cells in the meristem during the short steady-state growth period (12–36 h ASRP) averaging 13. The dynamics of meristem exhaustion obeyed Ivanov's model of the life span of cells in the meristem that states: if cell division is suppressed, half of the cells present in the meristem at a given time leave the meristem and start elongation during the period equal to the duration of the cell division cycle. It was deduced, on average, three to five cell division cycles in the meristem preceded its exhaustion. The lost meristem integrity can be related to only a few initial cells being found in the radicle. The cessation of meristematic activity in the primary-root apical meristem was directly related to the induction of lateral-root formation. Determinate primary-root growth can be thus viewed as a physiological root-tip decapitation that stops production of a signal inhibiting lateral-root primordia initiation. The time of lateral-root formation in S. gummosus and F. peninsulae was equal to or shorter than in agronomic mezophyte plants. Lateral roots also had determinate growth. The rapidity of root-system development and the ability to stop and to continue growth at any time under unfavorable and favorable conditions suggests the important role of determinate growth in seedling establishment of these Sonoran Desert species. Received: 13 December 1996 / Accepted: 6 January 1997     

Requirements for the light-stimulated degradation of stromal proteins in isolated pea (Pisum sativum L.) chloroplasts

Chloroplasts from 17-d-old pea leaves (Pisum sativum L.) wereisolated to elucidate the requirements for the light-induceddegradation of stromal proteins. The influence of electron transportthrough the thylakoids and the influence of ATP on protein degradationwere investigated. When chloroplasts were incubated in the light(45 µmol m^–2s^–1), glutamine synthetase, thelarge subunit of ribulose-1,5-bisphosphate carboxylase and glutamatesynthase were degraded, whereas phosphoribulokinase, ferredoxin-NADP⁺reductase and the 33 kDa protein of photosystem II remainedmore stable. Major protein degradation was not observed over240 mm in darkness. The electron transport inhibitor dichlorophenyldimethylureareduced protein degradation in the light over several hours,whereas dibromothymoquinone was less effective. Inhibiting theproduction of ATP with tentoxin or by destroying the     

Light-induced membrane potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum

Light transiently depolarizes the membrane of growing leaf cells. The ionic basis for changes in cell membrane electrical potentials in response to light has been determined separately for growing epidermal and mesophyll cells of the argenteum mutant of pea (Pisum sativum L.). In mesophyll cells light induces a large, transient depolarization that depends on the external Cl^– concentration, is unaffected by changes in the external Ca²⁺ or K⁺ concentration, is stimulated by K⁺-channel blockers tetraethylammonium (TEA⁺) and Ba²⁺, and is inhibited by 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU). In isolated epidermal tissue, light induces a small, transient depolarization followed by a hyperpolarization of the membrane potential. The depolarization is enhanced by increasing the external Ca²⁺ concentration and by addition of Ba²⁺, and is not sensitive to DCMU. Epidermal cells in contact with mesophyll display a depolarization resembling the response of the underlying mesophyll cells. The light-induced depolarization in mesophyll cells seems to be mediated by an increased efflux of Cl^– while the membrane-potential changes in epidermal strips reflect changes in the fluxes of Ca²⁺ and in the activity of the proton-pumping ATPase.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - CCCP carbonylcyanide m-chlorophenylhydrazone - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - LID _e light-induced depolarization in epidermal cells - LID _m light-induced depolarization in mesophyll cells - LIH light-induced hyperpolarization - TEA⁺ tetraethylammonium Ecotrans paper #43. This research was supported by National Science Foundation grants DCB-8903744 and MCB-9220110 to E.V.     

Growth and curvature in seedlings of Pisum sativum and an ageotropic mutant

In an attempt to explain the influence of gravity on the behaviour of ageotropic plant organs, a pea mutant (Pisum sativum ageotropum) and normal pea (Pisum sativum cv. Sabel) were examined. The mutant has a significantly lower germination rate (large seeds: 25%, small seeds: 10%) than normal pea seeds (55%). Removal of testa increased germination dramatically, the values obtained were 63 and 89%, respectively. Immediately after imbibition the mutant from which the testa had been removed, developed more slowly than normal pea seeds; after 28 h the difference in elongation rate between the two types was reversed. When continuously stimulated geotropically in the horizontal position the elongation in the mutant is larger than in the normal pea roots kept in the same position. During a 24 h period starting 48 h after imbibition the mutant root elongated 45.0 mm while the value for the normal pea root was 11.5 mm. The course of the geotropic curvature in roots of the two types has been followed during a period of 24 h. Normal pea roots develop an asymmetry in the extreme root tip region after 30 min of horizontal stimulation. After prolonged stimulation (exceeding 2 h) the asymmetry has disappeared and the curvature distributed over the entire growth region. When roots of normal pea are stimulated continuously at various angles, the optimum angle of geotropic response is 90° with decreasing responses in the order 135° (i.e. the root tip is pointing obliquely upward) and 45°. The presumed ageotropic behaviour of the mutant has only to a certain extent been confirmed in the present study. When stimulated at 135° a slight positive curvature developed; stimulation at 90° and 45° gave a slight negative curvature.     

Quantification of Apoplastic Potassium Content by Elution Analysis of Leaf Lamina Tissue from Pea (Pisum sativum L. cv Argenteum)

K⁺ content and concentration within the apoplast of mesophyll tissue of pea (Pisum sativum L., cv Argenteum) leaflets were determined using an elution procedure. Following removal of the epidermis, a 1 centimeter (inside diameter) glass cylinder was attached to the exposed mesophyll tissue and filled with 5 millimolar CaCl₂ solution (1°C). From time-course curves of cumulative K⁺ diffusion from the tissue, the amount of K⁺ of extracellular origin was estimated. Apoplastic K⁺ contents for leaves from plants cultured in nutrient solution containing 2 or 10 millimolar K⁺ were found to range from 1 to 4.5 micromoles per gram fresh weight, comprising less than 3% of the total K⁺ content within the lamina tissue. Assuming an apoplastic solution volume of 0.04 to 0.1 milliliters per gram fresh weight and a Donnan cation exchange capacity of 2.63 micromoles per gram fresh weight (experimentally determined), the K⁺ concentration within apoplastic solution was estimated at 2.4 to 11.8 millimolar. Net movement of Rb⁺ label from the extracellular compartment within mesophyll tissue into the symplast was demonstrated by pulse-chase experiments. It was concluded that the mesophyll apoplast in pea has a relatively low capacitance as an ion reservoir. Apoplastic K⁺ content was found to be highly sensitive to changes in xylem solution concentration.     

Reversal of blue light-stimulated stomatal opening by green light

Blue light-stimulated stomatal opening in detached epidermis of Vicia faba is reversed by green light. A 30 s green light pulse eliminated the transient opening stimulated by an immediately preceding blue light pulse. Opening was restored by a subsequent blue light pulse. An initial green light pulse did not alter the response to a subsequent blue light pulse. Reversal also occurred under continuous illumination, with or without a saturating red light background. The magnitude of the green light reversal depended on fluence rate, with full reversal observed at a green light fluence rate twice that of the blue light. Continuous green light given alone stimulated a slight stomatal opening, and had no effect on red light-stimulated opening. An action spectrum for the green light effect showed a maximum at 540 nm and minor peaks at 490 and 580 nm. This spectrum is similar to the action spectrum for blue light-stimulated stomatal opening, red-shifted by about 90 nm. The carotenoid zeaxanthin has been implicated as a photoreceptor for the stomatal blue light response. Blue/green reversibility might be explained by a pair of interconvertible zeaxanthin isomers, one absorbing in the blue and the other in the green, with the green absorbing form being the physiologically active one.     

Relative anion permeabilities and concentrations in leaf cells of Pisum sativum determined using electrical measurements and the Goldman equation

The electrical potential difference across the tonoplast ofpalisade mesophyll cells of leaves of Pisum sativum was 6.6± 0.8 mv (positive in the vacuole). The potential acrossthe plasmalemma depended on the particular anion accompanyingpotassium in the external solution. Assuming that the plasmalemmapotentials were diffusion potentials that could be analyzedusing the Goldman equation, the permeabilities of pyruvate,formate, .butyrate, acetate and bicarbonate into the cells werecalculated to be large compared with that of potassium, whilethe chloride permeability was relatively low. The upper limitsfor concentrations in the cytoplasm of palisade mesophyll cellsin pea leaves were as follows: potassium, 98 mM total monovalentorganic acids, 0.4 mM and bicarbonate, 0.2 mM. (Received March 3, 1971; )     

Signal Integration by ABA in the Blue Light-Induced Acidification of Leaf Pavement Cells in Pea (Pisum sativum L. var. Argenteum)

Leaf pavement cell expansion in light depends on apoplastic acidification by a plasma membrane proton-pumping ATPase, modifying cell wall extensibility and providing the driving force for uptake of osmotically active solutes generating turgor. This paper shows that the plant hormone ABA inhibits light-induced leaf disk growth as well as the blue light-induced pavement cell growth in pea (Pisum sativum L.). In the phytochrome chromophore-deficient mutant pcd2, the effect of ABA on the blue light-induced apoplastic acidification response, which exhibits a high fluence phase via phytochrome and a low fluence phase via an unknown blue light receptor, is still present, indicating an interaction of ABA with the blue light receptor pathway. Furthermore, it is shown that ABA inhibits the blue light-induced apoplastic acidification reversibly. These results indicate that the effect of ABA on apoplastic acidification can provide a mechanism for short term, reversible adjustment of leaf growth rate to environmental change.Key Words: ABA, apoplastic acidification, blue light, epidermal pavement cell growth, leaf growth, pea (Pisum sativum L.), signal integration     

Focusing of light by leaf epidermal cells

Leaf epidermal cells from a wide variety of plants focus light to surprisingly high levels. Using image analysis, the concentration and distribution of light was measured after it passed through epidermal cells within peels and epidermal cells attached to palisade cells in partially dissected leaves. In peels taken from Medicago sativa, Zea mays , and Impatiens sp., light was concentrated 15- to 20-fold by individual epidermal cells. When left attached to the mesophyll, which attenuated focusing by absorption and scattering, light was focused up to 5 times. The position of the focal spot beneath each epidermal cell was affected by the direction at which the light struck the cell. When the light was perpendicular to the leaf surface, individual focal spots fell beneath each epidermal cell. When the incident light was oblique, the focal spot shifted laterally and was positioned closer to the anticlinal cell wall. Focusing was observed when leaves were irradiated with collimated light but not with diffuse light. Focal lengths were relatively independent of wavelength within the visible region of the spectrum and there were only slight differences between focusing of blue vs red light. Epidermal lens properties can affect chlorophyll fluorescence and the photosynthetic performance of leaves. A survey of 47 species collected from a wide variety of habitats indicates that many plants have leaf epidermal cells with lens properties. The ability to measure epidermal focusing makes it possible to examine the adaptive and physiological significance of epidermal lens effects in plants.     

NaCl-stimulated proton efflux and cell expansion in sugar-beet leaf discs

The expansion of illuminated sugar-beet leaf discs floating on aqueous solutions is stimulated by 10 mM NaCl. During expansion, protons are pumped out of the cell and NaCl increases this proton flux by about 40%. The nett flux of K⁺ and Na⁺ into the discs was also evaluated. During the expansion period K⁺ decreases while Na⁺ increases markedly. The results indicate the existence of a sodium-stimulated proton pump which is active during cell enlargement.Abbreviations IAA indole-3-acetic acid - PEG polyethylene glycol     

Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

A temperature-sensitive nodulation mutant (sym 5) of Pisum sativum L.

Abstract. In peas ( Pisum sativum L.) homozygous for sym 5, nodulation has an unusual temperature dependence. These sym 5 mutants nodulate poorly at a root temperature of 20°C but nodulate better at 12°C. By lowering the root temperature of the sym 5 mutants from a lightroom temperature of 20/15°C to a constant 12°C, 8d after planting, the number of nodules can be further increased. A cool period (12°C) as short as 6h, early in the infection process, is sufficient to significantly increase nodulation of plants otherwise growing at 20/15°C. This temperature-sensitivity of nodulation is not due to a temperature induced change of a sym 5-related, 66-kD peptide but may involve accumulation of a gas in the rhizosphere.     

Isolation of a nitrate reductase deficient mutant of Pisum sativum by means of selection for chlorate resistance

Summary After EMS treatment of seeds of the Pisum variety Rondo a chlorate resistant mutant was isolated which showed a decrease in the in vitro activity of the enzyme nitrate reductase of roughly 95%. The mutation is monogenic and recessive. The mutant shows a decrease in protein content, and an increase in the amount of nitrate accumulated and in the activity of the enzyme nitrite reductase. On a liquid nutrient medium containing nitrate as the sole nitrogen source and in soil, the mutant grows very poorly due to necrosis of the leaves. On liquid medium containing ammonium, either with or without nitrate, growth is as good as that of the parent variety.     

Genetic interaction and mapping studies on the leaflet development (lld) mutant in Pisum sativum

In Pisum sativum, the completely penetrant leaflet development (lld) mutation is known to sporadically abort pinnae suborgans in the unipinnate compound leaf. Here, the frequency and morphology of abortion was studied in each of the leaf suborgans in 36 genotypes and in presence of auxin and gibberellin, and their antagonists. Various lld genotypes were constructed by multifariously recombining lld with a coch homeotic stipule mutation and with af, ins, mare, mfp, tl and uni-tac leaf morphology mutations. It was observed that the suborgans at all levels of pinna subdivisions underwent lld-led abortion events at different stages of development. As in leafblades, lld aborted the pinnae in leaf-like compound coch stipules. The lld mutation interacted with mfp synergistically and with other leaf mutations additively. The rod-shaped and trumpet-shaped aborted pea leaf suborgans mimicked the phenotype of aborted leaves in HD-ZIP-III-deficient Arabidopsis thaliana mutants. Suborganwise aborted morphologies in lld gnotypes were in agreement with basipetal differentiation of leaflets and acropetal differentiation in tendrils. Altogether, the observations suggested that LLD was the master regulator of pinna development. On the basis of molecular markers found linked to lld, its locus was positioned on the linkage group III of the P. sativum genetic map.