A glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls.

A particulate enzyme preparation made from epicotyls of 1-week-old etiolated pea (Pisum sativum) seedlings was shown to incorporate glucuronic acid from UDP-D-[U-14C]glucuronic acid into a hemicellulosic polysaccharide. Optimum conditions for the incorporation include the presence of Mn2+ ions at between 4 and 10 mmol/litre and a pH between 5 and 6. UDP-D-xylose at 1 mmol/litre allows incorporation to continue for at least 8 h. In its absence, the reaction stops within 30 min. Analysis of the product by partial and total acid hydrolysis, followed by paper chromatography or electrophoresis, indicates that the polysaccharide produced is a glucuronoxylan.     

The solubilization of a glucuronyltransferase involved in pea (Pisum sativum var. Alaska) glucuronoxylan synthesis.

A glucuronyltransferase involved in glucuronoxylan biosynthesis was obtained from the epicotyls of 1-week-old etiolated pea (Pisum sativum var. Alaska) seedlings and was solubilized in Triton X-100, a non-ionic detergent. The enzyme was inactivated by SDS and inhibited by Derriphat 160 and cholic acid. The enzyme was active in the presence of NN-dimethyldodecylanium-N-oxide, but was not solubilized by it. The stimulatory effect of UDP-D-xylose on the particulate and solubilized enzymes was the same, but the optimum Mn2+ concentration was lower for the solubilized enzyme, and the product formed by the solubilized enzyme has altered structure and solubility properties. Gel filtration of the solubilized enzyme on Sepharose CL-6B permitted partial separation of the stimulatory effect of UDP-D-xylose from the activity in the absence of UDP-D-xylose. The solubilized enzyme was more stable than the particulate enzyme and could be stored for 2 weeks at -20 degrees C without loss of activity.     

Distribution of xylosyltransferases and glucuronyltransferase within the Golgi apparatus in etiolated pea (Pisum sativum L.) epicotyls

Membranes from etiolated pea epicotyls were fractionated bydiscontinuous sucrose-density-gradient centrifugation into endoplasmicreticulum, Golgi apparatus and plasma membrane. The Golgi apparatuswas further fractionated on a shallow, continuous sucrose densitygradient into Golgi subfractions of low, medium and high density.Xylosyl- and glucuronyltransferases were both found to be concentratedin the Golgi apparatus. The glucuronyltransferase was concentratedin the low-density Golgi membranes, but xylosyltransferaseswere found in all three Golgi subfractions. The multiple locationof xylosyltransferases within the Golgi was found in both youngerand older regions of the epicotyl, and xylan was the major productof the xylosyltransferase in the low- and medium-density subfractions.In the presence of UDPglucose, xylose was also incorporatedinto xyloglucan, but this activity was concentrated in the highdensityGolgi membranes. Key words: Hemicellulose biosynthesis, xylan, xyloglucan, Golgi apparatus     

Protochlorophyllide forms in non-greening epicotyls of dark-grown pea (Pisum sativum)

Low-temperature fluorescence emission spectra of 6.5-day-old dark-grown epicotyls of pea ( Pisum sativum ) revealed the presence of protochlorophyll(ide). The upper part of the epicotyl contained 30% of the protochlorophyll(ide) content per fresh weight found in pea leaves, whereas the lower part contained 3%. Three discrete spectral forms of protochlorophyll(ide) were clearly distinguished after Gaussian deconvolution of fluorescence excitation and emission spectra. Adding the satellite bands of the Qy_(0-0) transitions (the emission vibrational (Emv) bands with correlated amplitudes, gave the following delineation: Ex439–Em629–Emv684, Ex447–Em636–Emv700 and Ex456–Em650–Emv728. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunodetection of whole tissue extracts of the epicotyl indicated the presence of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). Electron micrographs showed prolamellar bodies in at most 11 % of the plastid profiles of the epicotyl cells. These prolamellar bodies were smaller, and many of them showed less regular structure than those of the leaves. Taken together, the results indicate that the protochlorophyll(ide) in epicotyls is arranged in a different way than in leaves.     

Protochlorophyllide transformations and chlorophyll accumulation in epicotyls of pea (Pisum sativum)

Low-temperature fluorescence emission spectra of epicotyls of 6.5-day-old dark-grown seedlings of pea ( Pisum sativum L.) showed the dominance of short-wavelength protoch lorophyllide forms with emission maxima at 629 and 636 nm, respectively. The presence of long-wavelength protochlorophyllide with emission maxima around 650 nm was just detectable. Accordingly, irradiation with millisecond flashes gave a minute formation of chlorophyllide. The chlorophyll(ide) formation varied along the epicotyl. Irradiation with continuous light for 1.5 h resulted in an evident accumulation of chlorophyll(ide) in the upper part of the epicotyl. Only small amounts accumulated in the middle section. The conversion of protochlorophyllide to chlorophyllide was temperature dependent and almost arrested at 0°C. The chlorophyll(ide) formed had one dominating fluorescence peak at 681 nm. Irradiation for 24 h gave almost 100 times more chlorophyll in the upper part of the epicotyl than in the lower part. Electron micrographs from the upper part of the epicotyl irradiated for 6 h showed plastids with several developing thylakoids, while the plastids in the lower part of the epicotyl had only a few thylakoids. The dominance of short-wavelength protochlorophyllide forms indicated the presence of protochlorophyllide not bound to the active site of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33). The inability of the short-wavelength form to transform into chlorophyllide with flash light denotes a dislocation from the active site. The time and temperature dependence of the chlorophyll(ide) formation in continuous light indicates that a relocation is required of the short-wavelength protochlorophyllide before chlorophyllide formation can occur.     

Delayed chlorophyll accumulation and pigment photodestruction in the epicotyls of dark-grown pea (Pisum sativum)

A comparison was performed of the tetrapyrrole transformations that occur upon irradiation of epicotyl or leaves of dark-grown Pisum sativum L. (var. Zsuzsi, Hungary). High performance liquid chromatography analysis after continuous or flash-irradiation showed that the biosynthetic pathway from protochlorophyllide (Pchlide) to chlorophyll (Chl) a was markedly slowed down at the step of the reduction of geranylgeranyl(gg)-Chl to dihydrogeranylgeranyl (dhgg)-Chl in epicotyls, whereas phytyl-Chl was synthesized in leaves subjected to the same light treatments. Quantitative pigment analysis during continuous irradiations of different intensities also showed that significant Pchlide photodestruction occurred in epicotyls even under weak light. When both Pchlide and chlorophyllide and/or chlorophylls were present in epicotyls, Pchlide photodestruction was faster under 630-nm light than under 670-nm light, which indicates that this process is most efficiently promoted by Pchlide excitation. Pre-incubation of epicotyl segments with 10 m M ascorbate partly alleviated pigment photodestruction in white light. It is concluded that formation of photoactive Pchlide–Pchlide oxidoreductase complexes is important to prevent fast pigment photooxidation after Pchlide accumulation in the dark.     

Hollow heart of pea (Pisum sativum)

Book reviewed in this article: The Way Ahead in Plant Breeding. Proceedings of the Sixth Congress of Eucarpia. Edited by F. G. H. Lupton , G. Jenkins and R. Johnson . Basic Electron Microscope Techniques. By M. A. Hayat . The Logit Transformation (with special reference to its uses in bioassay). By W. D. Ashton . Families of Frequency Distributions. By J. K. Ord .     

Verticillium wilt of pea (Pisum sativum)

A wilt disease of garden pea (Pisum sativum) caused by Verticillium dahliae is described and the range of pathogenicity of the isolate investigated. It is pathogenic to potato, sweet pea, antirrhinum and broad bean and isolates of V. dahliae from potato, lucerne and sweet pea and V. albo-atrum from lucerne are pathogenic to pea. Since the most common disease symptoms, acropetal progression of chlorosis and necrosis of the leaves followed by premature defoliation are indistinguishable from natural senescence, it is probable that disease and senescence symptoms are confused in the field. The premature defoliation results in marked reduction in green leaf area, leaf dry weight and pod yield.     

Embryogenesis of the pea (Pisum sativum)

Summary The most striking internal feature of the suspensor cells inPisum is the abundant occurrence of a plastid containing spherical bodies consisting of intertwined bundles of tubules. These tubular complexes are not typical prolamellar bodies and they are not converted into grana. Cytochemical reactions indicate that they are proteinaneous. The participation of this plastid in the possible nutritional function of the suspensor is discussed but it is pointed out that critical experimental evidence is needed before the role of the suspensor and its contents in embryogenesis can be understood.Supported by a grant from the Australian Research Grants Committee.     

Light-induced wilting and its molecular mechanism in epicotyls of dark-germinated pea (Pisum sativum L.) seedlings

Possible mechanisms behind the light-induced wilting of dark-germinated pea (Pisum sativum L.) epicotyls were studied. Illumination with photosynthetically active radiation caused a fast turgor loss and wilting in the middle segments of the epicotyls accompanied by accumulation of water in the intercellular cavities. During this process, room temperature fluorescence emission spectra showed gradual bleaching of porphyrin-type pigments, which was lessened by incubating the epicotyls with excess ascorbate before illumination. Detection of singlet oxygen and lipid peroxidation products in the illuminated epicotyls suggested the occurrence of porphyrin-photosenzitized membrane damage as a cause of disordered water status and sequential wilting.     

GTP-binding proteins in etiolated epicotyls of Pisum sativum (Alaska) seedlings

Seven fractions of GTP-binding proteins separated by gel filtration of an extract of epicotyls of Pisum sativum seedlings were partially characterized. Seven fractions of GTP-binding proteins tentatively designated GP1 to GP7 had the capacity to be ADP-ribosylated by pertussis toxin. Pooled fractions of GP2 to GP7 showed Km values 2, 20, 50, 10, 3 and 1 nM, respectively. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented competitively in the presence of 0.1 mM GTP and also prevented in the presence of 0.1 mM ATP. Binding of [35S]GTP gamma S to the proteins produced a decrease in their molecular weights.     

Fast phototransformation of the 636 nm-emitting protochlorophyllide form in epicotyls of dark-grown pea (Pisum sativum)

The phototransformation of protochlorophyllide forms was studied in epicotyls of dark-germinated pea (Pisum sativum L. cv. Zsuzsi) seedlings. Middle segments were illuminated with white or 632.8 nm laser flash or continuous light at room temperature and at −15°C. At low light intensities, photoreduction could be distinguished from bleaching. 77 K fluorescence emission spectra were measured, difference spectra of illuminated and non-illuminated samples were calculated and/or the spectra were deconvoluted into Gaussian components. The 629 nm-emitting protochlorophyllide form, P629 (Pxxx where xxx is the fluorescence emission maximum), was inactive. For short-period (2–100 ms) and/or low-intensity (0.75–1.5 µmol m⁻² s⁻¹) illumination, particularly with laser light, the transformation of P636 into the 678 nm-emitting chlorophyllide form, C678 (Cxxx where xxx is the fluorescence emission maximum), was characteristic. This process was also found when the samples were cooled to −15°C. The transformation of P644 into C684 usually proceeded in parallel with the process above as a result of the strong overlap of the excitation bands of P636 and P644. The Shibata shift of C684 into a short-wavelength form, C675–676, was observed. Long-period (20–600 s) and/or high-intensity (above 10 µmol m⁻² s⁻¹) illumination resulted in the parallel transformation of P655 into C692. These results demonstrate that three flash-photoactive protochlorophyllide forms function in pea epicotyls. As a part of P636 is flash photoactive, its protochlorophyllide molecule must be bound to the active site of a monomer protein unit [Böddi B, Kis-Petik K, Kaposi AD, Fidy J, Sundqvist C (1998) The two short wavelength protochlorophyllide forms in pea epicotyls are both monomeric. Biochim Biophys Acta 1365: 531–540] of the NADPH:protochlorophyllide oxidoreductase (EC 1.3.1.33). Dynamic interconversions of the protochlorophyllide forms into each other, and their regeneration, were also found, which are summarized in a scheme.     

Boron alleviates aluminum toxicity in pea (Pisum sativum)

One important target of boron (B) deficiency and aluminum (Al) toxicity is cell wall. Thus we studied the hypothesis that B is capable of alleviating Al toxicity in pea (Pisum sativum). Short-term and prolonged Al exposure to pea roots at different B levels was carried out on uniform seedlings pre-cultured at a low B level. When seedlings with a low B level were supplied with or without B for 1 and 2 days before 24 h Al exposure, roots were longer while root diameter was thinner after B addition especially for 2 days even with exposure to Al; root elongation was inhibited while root diameter was enlarged by Al exposure. Callose induction by Al toxicity was higher with B added, but this was reversed after the removal of the cotyledons. Hematoxylin staining was lighter in the root tips given B, and Al content in the root tips and cell walls dropped after exposure to B. This indicates that B alleviated Al toxicity in the root tips during short-term Al exposure by decreasing Al binding in root cell walls. An increase in chlorophyll and biomass and reduced chlorosis were found at the higher level of B during prolonged Al treatment, which was coincided with the decreased Al contents, indicating that B alleviated Al toxicity to shoots. B supplementation alleviates some of the consequences of Al toxicity by limiting some Al binding in cell walls, resulting in less injury to the roots as well as less injury to the shoots.     

Distinct UV-A or UV-B irradiation induces protochlorophyllide photoreduction and bleaching in dark-grown pea (Pisum sativum L.) epicotyls

Photosynthesis Research - The effects of distinct UV-A and UV-B radiations were studied on etiolated pea (Pisum sativum L.) epicotyls. Emission spectra of the native protochlorophyll and...     

The protective effect of desferrioxamine on paraquat-treated pea (Pisum sativum)

Paraquat, a widely used herbicide, is photoreduced by photosystem I to the monovalent cation radical, which in turn, can react quickly and efficiently with molecular oxygen to produce superoxide anion radicals. In the presence of redox-active iron (or copper) superoxide radicals can serve as a source for the more active species such as hydroxyl radicals. The present sludv investigated the possible mediatory role of iron in paraquat to xicity. The results demonstrate that desferrioxamme (0–150μM) a highiy specific iron chelator, reduces the loss of proteins (by 34–69%) and lipid peroxidation (by 31–96%) in paraquat treated leaf cuts. Dcsferrioxamine also protects malate dehydrogenase (61–70%) hydroxvpyruvate reductase (54–100%), and Ca²⁺-dependent ATPase (25–34%) against the paraquat-induced loss of their activity. It also induces an increase in glutathione reductase activity (by 188%). These results, together with those from other experiments concerning the effect of desferrioxamine on paraquat uptake by the leaf cuts, suggest that the protection by desferrioxamine arises from its specific iron chelanon properties, and lead to the conclusion that nan-protein-bound and redoxactive forms of iron pluy a role in the manifestation of paraquat toxicity in plants.     

Regeneration of shoots from pea (Pisum sativum) hypocotyl explants

A sequential indole-3-acetic acid (IAA)-zeatin treatment was applied to Pisum sativum hypocotyl explants, resulting in shoot formation from 50% of the explants. Shoots were easily rooted and transplantable plants could be obtained in 3 months. The method has been applicable to the 5 cultivars tested. Histological examination of explants suggests the shoots to be of de novo origin, which would make the system suitable for transformation experiments.