Effects of light and acetate on the liberation of zoospores by a mutant strain ofChlamydomonas reinhardtii

In light-dark-synchronized cultures of the unicellular green algaChlamydomonas reinhardtii, release of zoospores from the wall of the mother cell normally takes place during the second half of the dark period. The recently isolated mutant ls, however, needs light for the liberation of zoospores when grown photoautotrophically under a 12 h light-12 h dark regime. The light-induced release of zoospores was found to be prevented by addition of the photosystem-II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Furthermore, light dependence of this process was shown to be abolished when the mutant ls was grown either photoautotrophically under a 14 h light-10 h dark regime or in the presence of acetate. Our findings indicate that the light-dependency of zoospore liberation observed in cultures of this particular mutant during photoautotrophic growth under a 12 h light-12 h dark regime might be attributed to an altered energy metabolism. The light-induced release of zoospores was found to be prevented by addition of cycloheximide or chloramphenicol, antibiotics which inhibit protein biosynthesis by cytoplasmic and organellar ribosomes, respectively. Actinomycin D, an inhibitor of RNA synthesis, however, did not affect the light-induced liberation of zoospores.Sporangia accumulate in stationary cultures of the mutant ls. Release of zoospores was observed when these sporangia were collected by centrifugation and incubated in the light after resuspension in fresh culture medium. Since liberation of zoospores was not observed after dilution of the stationary cultures with fresh culture medium, we suppose that components which interfere with the action of the sporangial autolysin are accumulated in the culture medium of the mutant ls.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Local and systemic changes in gene expression induced in tomato plants by wounding and by elicitor treatment

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants have been wounded to induce the accumulation of proteinase-inhibitor proteins (PI proteins) at the local site of injury and systemically in unwounded tissues. To determine the range of genes affected in the wound-response, polysomal mRNA has been isolated from the damaged leaves and from systemically responding leaves over a time-course of 2, 4, 10 and 24 h after wounding. Changes in the pattern of ³⁵S-translation products indicate that the events that occur at the local wound-site are different from those that occur systemically, both with respect to the number of genes that are regulated and the timing of their regulation. In order to compare the effects of wounding and an endogenous systemic signal generated at the wound-site with those of elicitor (proteinase-inhibitor-inducing factor, PIIF) treatment of excised plants, polysomal mRNA has also been isolated from leaves of plants over a time-course of 2, 4, 10 and 24 h after PIIF-treatment. Changes in the pattern of ³⁵S-translation products indicates that the events induced by PIIF resemble those induced by mechanical injury, rather than those induced by the endogenous systemic signal.Abbreviations IFF isoelectric focussing - PI proteins proteinase inhibitor proteins - PIIF proteinase-inhibitor-inducing factor - ssRubisco small subunit of ribulose-1,5-bisphosphate carboxylase     

Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of ¹⁴C-labelled efflux of 3-O-methyl-d-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K _1/2=7.9 mM) and its aglucon phloretin (K _1/2=84 μM); with both inhibitors, 3-O-methyl-d-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the d-enantiomers of neutral hexoses, including glucose (K _1/2=48 μM), 3-O-methyl-d-glucose (K _1/2=139 μM), and fructose (K _1/2=730 μM), but not by, for instance, d-allose, and l-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the d-glucose conformation.     

Calcium bridges are not load-bearing cell-wall bonds in Avena coleoptiles

I examined the ability of frozen-thawed Avena sativa L. coleoptile sections under applied load to extend in response to the calcium chelators ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) and 2-[(20bis-[carboxymethyl] amino-5-methylphenoxy)methyl]-6-methoxy-8-bis [carboxymethyl]aminoquinoline (Quin II). Addition of 5 mM EGTA to weakly buffered (0.1 mM, pH 6.2) solutions of 2(N-morpholino) ethanesulfonic acid (Mes) initiated rapid extension and wall acidification. When the buffer strength was increased (e.g. from 20 to 100 mM Mes, pH 6.2) EGTA did not initiate extension nor did it cause wall acidification. At 5 mM Quin II failed to stimulate cell extension or wall acidification at all buffer molarities tested (0.1 to 100 mM Mes). Both chelators rapidly and effectively removed Ca²⁺ from Avena sections. These data indicate that Ca²⁺ chelation per se does not result in loosening of Avena cells walls. Rather, EGTA promotes wall extension indirectly via wall acidification.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetraacetic acid - Quin II 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis(carboxymethyl)aminoquinoline - Mes 2(N-morpholino)ethanesulfonic acid     

The cell wall-plasmalemma interface in sieve tubes of barley

Both thick- and thin-walled sieve tubes in leaf-blade veins of Hordeum vulgare L. exhibit a distinct, electron-opaque inner wall layer after fixation in glutaraldehyde-osmium tetroxide and staining with uranyl acetate and lead citrate. This inner wall layer is thickest at the sieve plates and lateral sieve areas where it is permeated by a labyrinth of tubules formed by the plasmalemma. Along the lateral walls between sieve areas the inner wall layer apparently is penetrated by numerous microvilli-like evaginations of the plasmalemma, giving the cell wall-plasmalemma interface the appearance of a brush border. It is suggested that a similar brush-border-like structure may occur at the cell wall-plasmalemma interface of sieve elements in a wide variety of vascular plants.Abbreviation ER endoplasmic reticulum     

Sulphate influx in wheat and barley roots becomes more sensitive to specific protein-binding reagents when plants are sulphate-deficient

When young wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) plants were deprived of an external sulphate supply (-S plants), the capacity of their roots to absorb sulphate, but not phosphate or potassium, increased rapidly (derepression) so that after 3–5 d it was more than tenfold that of sulphate-sufficient plants (+S plants). This increased capacity was lost rapidly (repression) over a 24-h period when the sulphate supply was restored. There was little effect on the uptake of L-methionine during de-repression of the sulphate-transport system, but S input from methionine during a 24-h pretreatment repressed sulphate influx in both+S and-S plants.Sulphate influx of both+S and-S plants was inhibited by pretreating roots for 1 h with 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) at concentrations > 0.1 mol · m^-3. This inhibition was substantially reversed by washing for 1 h in DIDS-free medium before measuring influx. Longer-term pretreatment of roots with 0.1 mol·m^-3 DIDS delayed de-repression of the sulphatetransport system in-S plants but had no influence on+S plants in 3 d.The sulphydryl-binding reagent, n-ethylmaleimide, was a very potent inhibitor of sulphate influx in-S roots, but was much less inhibitory in +S roots. Its effects were essentially irreversible and were proportionately the same at all sulphate concentrations within the range of operation of the high-affinity sulphate-transport system. Inhibition of influx was 85–96% by 300 s pretreatment by 0.3 mol·m^-3 n-ethylmaleimide. No protection of the transport system could be observed by including up to 50 mol·m^-3 sulphate in the n-ethylmaleimide pre-treatment solution. A similar differential sensitivity of-S and+S plants was seen with p-chloromercuriphenyl sulphonic acid.The arginyl-binding reagent, phenylglyoxal, supplied to roots at 0.25 or 1 mol·m^-3 strongly inhibited influx in-S wheat plants (by up to 95%) but reduced influx by only one-half in+S plants. The inhibition of sulphate influx in-S plants was much greater than that of phosphate influx and could not be prevented by relatively high (100 mol·m^-3 sulphate concentrations accompanying phenylglyoxal treatment. Effects of phenylglyoxal pretreatment were unchanged for at least 30 min after its removal from the solution but thereafter the capacity for sulphate influx was restored. The amount of new carrier appearing in-S roots was far greater than in+S roots over a 24-h period.The results indicate that, in the de-repressed state, the sulphate transporter is more sensitive to reagents binding sulphydryl and arginyl residues. This suggests a number of strategies for identifying the proteins involved in sulphate transport.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenyl sulphonic acid     

Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L.

Nitrate reduction in roots and shoots and exchange of reduced N between organs were quantitatively estimated in intact 13-d-old seedlings of two-row barley (Hordeum vulgare L. cv. Daisengold) using the ¹⁵N-incorporation model (A. Gojon et al. (1986) Plant Physiol. 82, 254–260), except that NH ₊ ⁴ was replaced by NO _- ² . N-depleted seedlings were exposed to media containing both nitrate (1.8 mM) and nitrite (0.2 mM) under a light-dark cycle of 12:12 h at 20°C; the media contained different amounts of ¹⁵N labeling. Experiments were started either immediately after the beginning (expt. 1) or immediately prior to the end (expt. 2) of the light period, and plants were sampled subsequently at each light-dark transition throughout 36 h. The plants effectively utilized ¹⁵NO _- ³ and accumulated it as reduced ¹⁵N, predominantly in the shoots. Accumulation of reduced ¹⁵N in both experiments was nearly the same at the end of the experiment but the accumulation pattern in roots and shoots during each 12-h period differed greatly depending on time and the light conditions. In expt. 1, the roots accounted for 31% (light), 58% (dark), and 9% (light) of nitrate reduction by the whole plants, while in expt. 2 the contributions of the root were 82% (dark), 20% (light), and 29% (dark), during each of the three 12-h periods. Xylem transport of nitrate drastically decreased in the dark, but that of reduced N rather increased. The downward translocation of reduced ¹⁵N increased while nitrate reduction in the root decreased, whereas upward translocation decreased while nitrate reduction in the shoot increased. We conclude that the cycling of reduced N through the plant is important for N feeding of each organ, and that the transport system of reduced N by way of xylem and phloem, as well as nitrate reduction by root and shoot, can be modulated in response to the relative magnitude of reduced-N demands by the root and shoot, with the one or the other predominating under different circumstances.Symbols Anl accumulation of reduced ¹⁵N from ¹⁵NO _- ³ in ¹⁴NO _- ³ -fed roots of divided root system - Ar accumulation in root of reduced ¹⁵N from ¹⁵NO _- ³ - As accumulation in shoot of reduced ¹⁵N from ¹⁵NO _- ³ - Rr ¹⁵NO _- ³ reduction in root - Rs ¹⁵NO _- ³ reduction in shoot - Tp translocation to root of shoot-reduced ¹⁵N from ¹⁵NO _- ³ in phloem - Tx translocation to shoot of root-reduced ¹⁵N from ¹⁵NO _- ³ in xylem     

Molecular characterization of the diurnal/circadian expression of the chlorophyll a/b-binding proteins in leaves of tomato and other dicotyledonous and monocotyledonous plant species

Diurnal oscillations of steady-state mRNA levels encoding the chlorophyll a/b-binding proteins were monitored inLycopersicon esculentum, Glycine max, Phaseolus vulgaris, P. aureus, P. coccineus, Pisum sativum, Sinapis alba, Hordeum vulgare, Triticum aestivum andZea mays. In these plant speciescab mRNA accumulation increases and decreases periodically indicating i) that the expression of the genes for chlorophyll a/b-binding proteins (cab genes) is controlled by a circadian rhythm, and ii) that the rhythm is widely distributed among monocotyledonous and dicotyledonous plant species. A detailed characterization of the pattern ofcab mRNA expression in tomato leaves shows that the amplitude of the oscillation is dependent on i) the developmental stage of the leaves, ii) the circadian phase and duration of light and iii) the circadian phase and duration of darkness. In addition to the chlorophyll a/b-binding proteins, genes coding for other cellular functions were examined for cyclic variations of their mRNA levels. The analysis includes genes involved in i) carbon metabolism (e.g. phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, alpha amylase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase), ii) photosynthesis (large and small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, Q_B-binding protein, reaction-center protein of photosystem I) and iii) other physiological or morphological reactions (e.g. ubiquitin, actin). However, no periodic fluctuation pattern was detected for the mRNA levels of these genes in tomato and maize leaves.     

Patch clamping corn protoplasts

Summary Cell wall regeneration around protoplasts from Black Mexican Sweet corn suspension cells has been observed using scanning electron microscopy. A coherent array of cellulose microfibrils can be seen around protoplasts two hours after they have been isolated. This array does not form in the presence of 15 mg/l Congo Red. The frequency and electrical resistance of seals made between patch clamp pipettes and the plasmalemma around corn protoplasts is not significantly affected by the presence or absence of these fibrils (p₀=0.75); it remains relatively low. Some single channel records from BMS corn protoplasts are shown.