Soluble auxin-binding proteins in pea epicotyls

Auxin-binding proteins, have been identified in the soluble cytoplasrnic protein fraction of etiolated pea epicotyls, Pisum sativum L., cv. "Dippes Gelbe Victoria". The binding is specific for the auxins NAA, IAA and 2,4-D with a K_D in the range of 0.1–0.4 μ M . Moreover, the binding is competitive, sensitive to digestion by proteinase and shows linearity with the protein content of the assay mixture. The binding proteins appear to be very labile, since repeated freezing and thawing destroys specific binding. No clear pH-optimum could be detected in the physiological pH-range 5.5–8.0, but the binding was doubled at pH 8.0 compared to pH 5.5–7.0.     

Glycanase activities associated with cell walls ofCicer arietinum L. epicotyls

The effect of the proteins extracted with 1 M LiCl from theCicer arietinum etiolated epicotyl cell walls on the degradation of polysaccharides extracted with alkali was studied. The hemicellulosic polysaccharides were fractionated into three fractions extracted with 4 % KOH, 4 % KOH containing 8 M urea, and 24 % KOH. The protein extract showed exo-glycanase activity against all three hemicellulosic fractions whilst endo-glycanase activity was shown mainly against the hemicellulosic polysaccharides extracted with 4% KOH.     

Autolysis of the cell wall. Its possible role in endogenous and IAA-induced growth in epicotyls of Cicer arietinum

Indoleacetic acid (IAA), a factor that induces growth in epicotyls of cicer arietinum L. cv. Castellana, increases the autolytic capacity of the cell walls by 50%, suggesting that autolysis is related to the processes of cell wall loosening that accompany growth. IAA promotes an increase in the specific activities of the enzymes involved in autolysis, mainly α-galactosidase (EC 3.2.1.22). This relationship autolysis-growth. was also observed in a study of the autolytic capacity of cell walls from regions of the epicotyl with different growth capacity. The sugars released and the level of enzymatic protein were higher in the subapical region that towards the base.     

Behaviour of the cell walls of Aspergillus niger during the autolytic phase of growth

Abstract Autolysis of Aspergillus niger cultures has been studied. The stability of the cell wall fraction was followed throughout a 57-day period of autolysis. The results indicated that both a persistence of existing walls and an extra wall deposition due to continued synthesis of walls or wall components during the first 12 days of autolysis seem to take place.     

Changes in concentration of endogenous growth inhibitors during growth recovery of dwarf pea seedlings

In order to clarify the role of endogenous growth inhibitors A-2α and A-2β in a dwarf pea plant, red light (emission peak 657 nm) treated, 9-d-old seedlings of dwarf pea (Pisum sativum L. cv. Progress No. 9) were transferred to darkness, and the resulting changes in growth rate and concentrations of A-2α and A-2β were monitored. The growth rate of the epicotyls increased, and the concentration of the inhibitors in the epicotyls decreased, according to sigmoidal time courses. The relationship between the logarithms of the concentration of the inhibitors and the corresponding growth rate was linear. These results suggest that A-2α and A-2β, may play an important role in the growth recovery process of the dwarf pea cultivar after termination of red light irradiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ascorbic acid effect on the onset of cell proliferation in pea root

The ability of ascorbic acid to induce cell proliferation of non-cycling cells was investigated in quiescent embryo root of Pisum sativum L. cv. Lincoln, as well as in the active plantlet root meristem, where a minor portion of the cells is non-proliferating. Quiescent embryo cells speeded up the G₀–G₁ transition during germination in the presence of ascorbic acid. In addition, proliferating cells present in the root tip of 3-day-old plantlets, arrested at the G₁/S boundary by hydroxyurea, resumed the cycle earlier than the control, when treated with ascorbic acid. In contrast, ascorbic acid was unable to induce the proliferation of non-cycling cells present in the active meristem. Therefore, these data suggest that the ability of ascorbic acid lo induce cell proliferation depends on the physiological status of the cell. In particular the data indicate that ascorbic acid is involved in cell proliferation as a factor necessary to enable already competent cells to progress through the cell cycle phases, but not as a factor able to induce non-competent cells to overcome proliferation arrest.     

Enzymatic control of the accumulation of verbascose in pea seeds

Verbascose, the pentasaccharide of the raffinose family of oligosaccharides, consists of galactose units joined to sucrose. In pea (Pisum sativum) seeds, the content of verbascose is highly variable. In a previous study on a high‐verbascose pea cultivar, the present authors have demonstrated that verbascose is synthesized by a multifunctional stachyose synthase (EC 2.4.1.67), which utilizes raffinose as well as stachyose as a galactosyl acceptor. Herein the results of a study of the cloning and functional expression of stachyose synthase from the low‐verbascose genotype SD1 are reported and it is demonstrated that this line contains a protein with a reduced ability to synthesize verbascose. Analysis of seeds from seven pea lines revealed a positive correlation between verbascose synthase activity and verbascose content. Among these genotypes, only the SD1 line showed low verbascose synthase activity when the data were normalized to stachyose synthase activity. These results suggest that differences in the level of verbascose synthase activity could be caused by mutations in the stachyose synthase gene as well as by variation in the amount of the protein. The lines were also analysed for activity of α‐galactosidase, a catabolic enzyme that could limit the extent of verbascose accumulation. No relationship was found between α‐galactosidase activity and the amount of raffinose family oligosaccharides.     

Effect of growth temperature on the composition of phenols in pea roots

It was shown that, in pea (Pisum sativum L.) roots, flavans are a dominating component of the soluble phenol fraction. In plants grown at low temperature (8°C), flavan content during the early growth phase was lower than in plants grown at 22°C, but later it increased and was by 40% higher than in plants grown at 22°C. Total phenol content in the two treatments differed insignificantly. Low growth temperature decreased the content of some phenolic compounds in pea seedling roots.     

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.     

Stimulation of Pisum sativum epicotyl elongation by gibberellin and auxin. – Different effects of two hormones on osmoregulation and cell walls

The possible involvement of auxin in the action of gibberellin in stimulating cell elongation was examined by comparing the effects of gibberellic acid (GA) and IAA on the growth, osmoregulation and cell wall properties of the Alaska pea ( Pisum sativum L. cv. Alaska) subhook. Both GA and IAA stimulated cell elongation in the subhook region of derooted cuttings. Cotyledon excision decreased the stimulating effect of GA on the growth of the subhook region, but did not affect that of IAA. As the subhook region elongated, the osmotic potential of the cell sap and the total amount of osmotic solutes increased. Cotyledon excision accelerated the increase in the osmotic potential and suppressed the accumulation of osmotic solutes. In cuttings with cotyledons. GA partly counteracted the increase in the osmotic potential and substantially promoted the accumulation of osmotic solutes. On the other hand, in cuttings without cotyledons. GA did not affect the change in the osmotic potential although it slightly promoted the accumulation of osmotic solutes. IAA accelerated the increase in the osmotic potential, but did not affect the accumulation of osmotic solutes. IAA enhanced the extensibility of the cell wall, while GA did not affect it. These results suggest that at least in the Alaksa pea subhook region. GA does not stimulate cell elongation by affecting the level of auxin.     

Phosphoenolpyruvate carboxykinase in developing pea seeds is associated with tissues involved in solute transport and is nitrogen-responsive

The aim of this work was to investigate the occurrence of phosphoenolpyruvate carboxykinase (PEPCK) in developing pea (Pisum sativum) seeds in relation to their nitrogen supply. PEPCK was present throughout development, with the peak of PEPCK protein and activity in the seed coat and cotyledons preceding protein accumulation in the cotyledons. It showed a different developmental pattern from enzymes involved in amino acid metabolism (phosphoenolpyruvate carboxylase, glutamine synthetase and glutamate dehydrogenase). Immunolocalization showed that PEPCK was present in parts of the developing seed that are involved in the transport and metabolism of assimilates. Early in development, it was associated with the inner integument of the ovule, the endospermic cytoplasm and the outer cells of the embryo. In the middle of development, around the peak of activity, PEPCK was abundant at the outer surface of the developing cotyledons, in the embryonic axis and in the vasculature of the seed coat. Later in development, PEPCK was associated with the embryonic leaf primordia and meristem and cortex of the radicle. PEPCK protein was strongly induced in vitro in the seed coat by nitrate, ammonium and asparagine, in the cotyledons by asparagine and in planta by the supply of nitrogen, which led to an increase in asparagine secretion by empty seed coats. It is suggested that PEPCK is involved in the metabolism of nitrogenous solutes in developing pea seeds.     

The presence of a p53-like protein during pea seed maturation and germination

ABSTRACT

The mechanisms that allow monitoring of DNA damage and the activation of repair systems in plants are poorly known. In mammalian cells the tumor suppressor protein p53 plays an important role in the checkpoint pathway induced by DNA damage. In this work, we investigated the presence and distribution of the p53-like protein in pea root tip nuclei and its role during early germination in relation to DNA damage. In pea seed, PFGE and TdT assays show that DNA fragmentation occurs during maturation and dry seed storage, and that this DNA fragmentation is repaired at the beginning of germination before the onset of proliferation. In the same seeds, the p53-like protein was found during maturation and germination. Immunoblotting characterization of this protein led to the identification of a single specific protein of about 94 kDa, more abundant at the beginning of the hydration process than in actively cycling cells. Furthermore, the p53-like protein revealed different nuclear distribution patterns, probably in relation to the formation of DNA fragments in dry seeds, and to the reactivation of repair mechanisms during early germination. These data suggest that the presence of a p53-like protein in quiescent or proliferating pea embryo cells is related to DNA damage, and serves for the maintenance of genetic information and the development of normal seedlings.     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.     

The effects of nitrate on the enzymes involved in nitrogen assimilation in nodulated pea roots

Pea Plants ( Pisum sativaum L. ev. Little Marvel) were grown in N-free medium and when well nodulated (28 days) were supplied for 8 days with nitrate or ammonium. Over the 8 days of nitrate treatment, total amino and amide N in sap declined, and the proportion of aspartate relative to the other amino acids increased. After 8 days of treatment, nitrogenase (EC 1.18.2.1) activity in nitrate-treated plants declined to about 30% of the activity in controls even though nodules were not directly in contact with nutrient solution. Nitrogenase activity was also decreased by the addition of ammonium chloride (10 m M ). With addition of nitrate or ammonium. clear signs of senescence began to show in the nodules after 4 days. Nitrate reductase (EC 1.6.6.1) activity was induced in roots by nitrate, but decreased sharply in nodules. In response to nitrate addition, newly formed root tissues showed 3- to 5-times higher glutamine synthetase (GS. EC 6.3.1.4) activity than newly formed tissues of control plants, expressed on a protein or weight basis. In complementary experiments, when ammonium salts were used instead of nitrates, the increase in GS activity was significantly lower. GS activity decreased in nodules of treated plants and total extractable protein was 3 times lower in nodules of nitrate-treated plants than in controls at day 8 of treatment.     

Neutral growth inhibitors in light-grown dwarf pea shoots –separation, identification and growth regulation

To correlate endogenous growth inhibitors of dwarf pea to its dwarfism a thorough search of growth inhibitors was made in the neutral fraction of an acetone extract from the shoots of light-grown seedlings of Pisum sativum L. cvs Progress No 9 and Alaska. From both cultivars six inhibitors were separated and named A-1, A-2, A-3, B-1, B-2 and B-3 based on their order of elution from the silica gel column. Their contents as determined by cress root bioassay were: in cv. Progress 0.40, 16.5, 6.36, 1.02, 0.11 and 0.10, and in cv. Alaska 0.33, 2.35, 3.51, 0.95, 0.10 and 0.09 cress units (g fresh weight)⁻¹. Their contributions to growth regulation of the relevant pea plants were estimated as the products of the above-stated contents times the ratios of the specific activities of each standard sample in the cress roots and the relevant pea cultivars, and they were; in cv. Progress A-2, 5.44 and A-3, 2.10, and in cv. Alaska A-2, 0.68 and A-3, 0.88, those of A-2 and A-3 constituting more than 90% of the total contribution of the six inhibitors in either cultivar. The great differences in the content and contribution to growth regulation of A-2 and A-3 between the two cultivars suggest that the higher contents of and greater responsiveness to the two inhibitors in cv. Progress may be causes of dwarfism of this cultivar. A-l and A-3 were spectroscopically identified with pisatin and a mixture of cis, trans– , and trans, trans xanthoxins.     

Galactose loss and fruit ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls

Cell wall material (CWM) was prepared from nine fruit species at two ripening stages (unripe and ripe) and extracted sequentially with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na₂CO₃ and 4 M KOH. Each solubilised fraction and the CWM-residue remaining after 4 M KOH extraction was analysed for non-cellulosic sugar composition. A common pattern of distribution for polyuronide and pectin-associated neutral sugar was observed for all unripe fruit. Most polyuronide was extracted in the CDTA/Na₂CO₃ fractions while 70–93% of the neutral sugar was located on pectic polysaccharides in the 4 M KOH-soluble and CWM-residue fractions. During ripening, most of the galactose was lost from pectic polysaccharides in the CWM-residue. Partial solubilisation of these polysaccharides was achieved by treating the CWM-residue with endopolygalacturonase. The solubilised polysaccharides were separated into two fractions by ion-exchange chromatography. One of these contained polysaccharides with average molecular weights of 400 kDa or larger and consisted of between 70 and 90% arabinogalactan. The galactosyl residues were 80–90% β-1→4 linked, indicating largely unbranched side-chains. The arabinosyl residues were distributed among terminal, 3-, 5-, 2,5-, and 2,3,5-linked residues, indicating a highly ramified structure. The results are discussed with regard to the relationship between pectin solubilisation and galactose loss and their respective contribution to fruit softening. Received: 28 January 1997 / Accepted: 11 March 1997     

Non-photosynthetic mechanisms of growth reduction in pea (Pisum sativum L.) exposed to UV-B radiation

Pisum sativum cv. Guido grown under controlled environment conditions was exposed to either low or high UV-B radiation (2·2 or 9·9 kJ m^–2 d^–1 plant-weighted UV-B, respectively). Low or high UV-B was maintained throughout growth (LL and HH treatments, respectively) or plants were transferred between treatments when 22 d old (giving LH and HL treatments). High UV-B significantly reduced plant dry weight and significantly altered plant morphology. The growth and morphology of plants transferred from low to high UV-B were little affected, when compared with those of LL plants. By contrast, plants moved from high to low UV-B showed marked increases in growth when compared with HH plants. This contrast between HL and LH appeared to be related to the effect of UV-B on plant development. Exposure to high UV-B throughout development consistently reduced leaf areas. In fully expanded leaves there was no significant UV-B effect on cell area and reduced leaf area could be attributed to reduced cell number, suggesting effects on leaf primordia. Further reductions in the leaf area of younger leaves were the result of the slower development rate of plants grown at high UV-B, which also resulted in significant reductions in leaf number.     

Effect of temperature on electron transport activities of glutaraldehyde-fixed pea thylakoids

Temperature-induced changes in Hill activity of glutaraldehyde-fixed pea ( Pisum sativum L. cv. Alaska) thylakoids have been examined. Using ferricyanide as electron acceptor, a temperature-induced change occurred at ca 12–14°C for both control and fixed thylakoids. In contrast to the controls, fixed thylakoids not only showed a change in slope of the Arrhenius plots but also a discontinuity which has not been observed in previous studies. A drop in activity coincided with the decrease in slope: the extent of the reduction depended on the concentration of glutaraldehyde used for fixation. Using a lipophilic electron acceptor, a temperature-induced change also occurred at 12–14°C, but there was no reduction in activities of fixed thylakoids at temperatures above the change in slope.
The results indicate that a temperature-induced change in fixed thylakoids restricts the access of ferricyanide to its reductant(s) in the membrane but that fixation does not affect the temperature-induced change per se. The results confirm that temperature has a general effect on the functioning of thylakoid membranes. The data demonstrate that calculations of the extent of inhibition by glutaraldehyde of Hill activity with ferricyanide should take into account the temperature at which assays are performed.     

Inhibition of cell wall autolysis and auxin-induced elongation of Cicer arietinum epicotyls by β-galactosidase antibodies

Polyclonal antibodies were raised in response to βIII-galactosidase purified from cell wall of Cicer arietinum epicotyls. The antibody preparation generated, bound to βIII protein giving a major protein band in the zone corresponding to M_r 45 000, the molecular mass previously estimated for βIII-galactosidase. These antibodies clearly suppress autolytic reactions in isolated walls of Cicer arietinum epicotyl segments, while the preimmune serum had no effect on autolytic reaction. The results strongly support the idea that the autolytic degradation of the cell wall is carried out by the βIII-galactosidase.
The antibodies against β-galactosidase were also able to inhibit cell wall hydrolysis mediated by both total cell wall protein extracted by LiCl and cell wall hydrolysis mediated by βIII-galactosidase.
Since autolysis is thought to be related to the process of cell wall loosening, the effects of the antibodies against the autolytic enzyme was also tested on epicotyl growth. β-galactosidase antibodies consistently inhibited IAA-induced elongation.     

The rates of shoot and root growth in intact plants of pea mutants in leaf morphology

Isogenic lines of pea (Pisum sativum L.) with the genetically determined changes in leaf morphology, afila (af) and tendril-less (tl), were used to study the relationship between shoot and root growth rates. The time-course of shoot and root growth was followed during the pre-floral period in the intact plants grown under similar conditions. The af mutation produced afila leaves without leaflets, whereas in the case of the tl mutations, tendrils were substituted with leaflets, and acacia-like leaves were developed. Due to the changes in leaf morphology caused by these mutations, pea genotypes differed in leaf area: starting from day 7, the leaf area was lower in the af plants and larger in the tl plants as compared to the wild-type plants. Such divergence was amplified in the course of plant development and reached its maximum immediately before the transition to flowering. Plants of isogenic lines did not notably differ in stem surface areas. In spite of significant difference in total leaf area, the wild type and tl plants did not differ in leaf dry weight. Starting from leaf 9, the af plants lagged behind two leaflet-bearing genotypes (wild type and tl) in leaf dry weight, whereas stem dry weight was similar in the wild type and tl forms and slightly lower in the af plants. Root dry weights were practically similar in the wild type and tl plants until flowering. The reduction of leaf area in the af plants drastically reduced root dry weight. In other words, the latter index was related to the total weight and total area of leaves and stems. The correlation analysis demonstrated an extremely low relationship between leaf and stem area and dry weight and those of roots early in plant development (when plants develop five to seven leaves). Later, immediately before flowering (nine to eleven leaves), root weight was positively related to leaf weight and area; however, stem area and root weight did not correlate. Thus, in three genotypes (wild type, af, and tl), at the end of their vegetative growth phase, leaf and root biomass accumulated in proportion, independently of leaf area expansion.