Viability of Cururbita pepo pollen: biophysical and structural data

During ageing of the short-lived pollen grains of Cucurbita pepo L., water loss was examined in relation to viability using biophysical (¹H-nuclear magnetic resonance, NMR) and cytological methods (fluorochromatic reaction test, freezefracture and scanning electron microscopy). A semi-logarithmic representation of the pollen weight loss demonstrated the complexity of the dehydration process. A the study of proton loss using ¹H-NMR indicated that two major releases water of had taken place, each with different flux rates. Pulse ¹H-NMR experiments showed the occurrene of non-exponential signal decay as a function of time, indicating the existence of different fractions of water in a pollen grain sample. These fractions leave the pollen grain at different times during pollen dehydration, and one of them (that of the so-called vital water) can be related to pollen viability. The quantity of protons giving a signal during pulse ¹H-NMR experiments was very low when the pollen grains were judged to be dead according to the fluorochromatic test. Freeze-fracture replicas of these dead pollen grains (less than 25% water content) showed that the plasma membrane had become detached from the intine surface; this ultrastructural feature might therefore be involved in the loss of pollen viability.Abbreviations A initial amplitude of the NMR signal - A₂ quantity of water charcterized by T_2-2 - A₅ quantity of water characterized by T_2–5 - FCR fluorochromatic reaction - NMR nuclear magnetic resonance - T₂ transverse relaxation time - T_2-2 T₂ measured with 2 ms between each pulse of radiofrequency - T_2–5 T₂ measured with 5 ms between each pulse of radiofrequency     

Different routes for integral protein insertion into Ricinus communis protein-body and glyoxysome membranes

Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB antiserum to integral protein-body membrane proteins - anti-G antiserum to integral glyoxysomal membrane proteins - anti-L antiserum to alkaline lipase - ER endoplasmic reticulum - M_r relative molecular mass - mRNA poly(A)-rich messenger RNA - PAGE polyacrylamide gel electrophoresis - poly(A) polyadenylic acid - SDS sodium dodecyl sulphate     

Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

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.     

Radial transport of water across cortical sleeves of excised roots ofZea mays L.

The stationary radial volume flows across maize (Zea mays L.) root segments without steles (sleeves) were measured under isobaric conditions. The driving force of the volume flow is an osmotic difference between the internal and external compartment of the root preparations. It is generated by differences in the concentrations of sucrose, raffinose or polyethylene glycol. The flows are linear functions of the corresponding osmotic differences ( ) up to osmotic values which cause plasmolysis. The straight lines obtained pass through the origin. No asymmetry of the osmotic barrier could be detected within the range of driving forces applied ( =±0.5 MPa), corresponding to volume-flow densities of j_{v, s}=±7·10^–8 m·s^–1. Using the literature values for the reflection coefficients of sucrose and polyethylene glycol in intact roots (E. Steudle et al. (1987) Plant Physiol.84, 1220–1234), values for the sleeve hydraulic conductivity of about 1·10^–7 m·s^–1 MPa^–1 were calculated. They are of the same order of magnitude as those reported in the literature for the hydraulic conductivity of intact root segments when hydrostatic pressure is applied.Abbreviations and symbols a _s outer surface of sleeve segment - c concentration of osmotically active solute - j _{v, s} radial volume flow density across sleeve segment - Lp_s hydraulic conductivity of sleeves - Lp_r hydraulic conductivity of intact roots - _N thickness of Nernst diffusion layer - reflection coefficient of root for solute - osmotic value of bulk phase - osmotic coefficient     

Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.     

Chromatographic and immunological evidence that chloroplastic and cytosolic pea (Pisum sativum L.) NADP-isocitrate dehydrogenases are distinct isoenzymes

Two NADP-isocitrate dehydrogenase isoenzymes designated as NADP-IDH₁ and NADP-IDH₂ (EC 1.1.1.42) were identified in pea (Pisum sativum) leaf extracts by diethylaminoethylcellulose chromatography. The predominant form was found to be NADP-IDH₁ while NADP-IDH₂ represented only about 4% of the total leaf enzyme activity. These enzymes share few common epitopes as NADP-IDH₂ was poorly recognized by the specific polyclonal antibodies raised against NADP-IDH₁, and as a consequence NADP-IDH₂ does not result from a post-translational modification of NADP-IDH₁. Subcellular fractionation and isolation of chloroplasts through a Percoll gradient, followed by the identification of the associated enzymes, showed that NADP-IDH₁ is restricted to the cytosol and NADP-IDH₂ to the chloroplasts. Compared with the cytosolic isoenzyme, NADP-IDH₂ was more thermolabile and exhibited a lower optimum pH. The data reported in this paper constitute the first report that the chloroplastic NADP-IDH and the cytosolic NADP-IDH are two distinct isoenzymes. The possible functions of the two isoenzymes are discussed.Abbreviations BSA bovine serum albumin - DEAE diethylaminoethyl - NADP-IDH NADP-isocitrate dehydrogenase - NADP-IDH₁ cytosolic NADP-IDH - NADP-IDH₂ chloroplastic NADP-IDH     

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     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate