Rapid alterations in growth rate and electrical potentials upon stem excision in pea seedlings

Excision of the epicotyl base of pea (Pisum sativum L.) seedlings in air results in a fast drop in the growth rate and rapid transient membrane depolarization of the surface cells near the cut. Subsequent immersion of the cut end into solution leads to a rapid, transient rise in the epicotyl growth rate and an acropetally propagating depolarization with an amplitude of about 35 mV and a speed of approx. 1 mm · s^–1. The same result can be achieved directly by excision of the pea epicotyl under water. Shape, amplitude and velocity of the depolarization characterize it as a slow-wave potential. These results indicate that the propagating depolarization is caused by a surge in water uptake. Neither a second surge in water uptake (measured as a rapid increase in growth rate when the cut end was placed in air and then back into solution) nor another cut can produce the depolarization a second time. Cyanide suppresses the electrical signal at the treated position without inhibiting its transmission through this area and its development in untreated parts of the epicotyl. The large depolarization and repolarization which occur in the epidermal and subepidermal cells are not associated with changes in cell input resistance. Both results indicate that it is a transient shut-down of the plasma-membrane proton pump rather than large ion fluxes which is causing the depolarization. We conclude that the slow wave potential is spread in the stem via a hydraulic surge occurring upon relief of the negative xylem pressure after the hydraulic resistance of the root has been removed by excision.Abbreviations and Symbols GR growth rate - P_x xylem pressure - R_in cell input resistance - SWP slow wave potential - V_m membrane potential - V_s surface potential This work was supported by grants to D.J.C. from the National Science Foundation and the U.S. Department of Energy.     

Responses of grass pea seedlings to salinity stress in in vitro culture conditions

Physiological and molecular mechanisms of adaptation to abiotic stresses of grass pea (Lathyrus sativus L.) are still poorly understood. Responses of four genotypes of grass pea to salinity stress in tissue culture conditions were investigated at early seedling growth stages. Salinity stress was induced in the agar media by adding 0, 50, 100 and 200 mM of NaCl. Germination and seedling emergence percentage was not significantly affected by 50 and 100 mM of NaCl. However, NaCl in 200 mM concentration lowered level of these parameters. Generally, exposure to NaCl stress significantly reduced length of grass pea seedling organs (root and shoot) but did not influence the content of dry weight in shoots and increased it in the roots in two cases. Increasing salt concentration decreased integrity of cellular membranes both in root and shoot tissues. Higher accumulation of phenolic compounds and significant changes in activity of antioxidant enzymes (peroxidase and catalase) were observed in the roots but not in the shoots. Similarly, the content of proline increased mostly in the roots from moderate (100 mM) salinity conditions. Adverse conditions did not resulted in alterations in photosynthetic pigments content of any tested genotypes. The better performance of shoots than roots may result from in vitro conditions in which experiments were conducted.

     

UDPG:Sterol glucosyltransferase in etiolated pea seedlings

Sterol UDPglucose glucosyltransferase was located predominantly in the axis tissue of etiolated pea seedlings. During the first 11 days of growth the activity reached a peak in the axis tissue after seven days. Centrifugation of tissue homogenates showed the cell fraction sedimenting between 13000 nd 25000 g to have the highest specific activity and also the bulk of the total activity. Sitosterol is the major free sterol of this fraction and cholesterol is a trace component. The composition of the aglycones of the isolated steryl glycosides shows cholesterol to be the major sterol. Although exhibiting no metal ion requirement, the enzyme is stimulated by Ca²⁺ and Mg²⁺, partially inhibited by EDTA and EGTA and completely inhibited by Zn²⁺. The membranous nature of the enzyme is manifested by its stimulation by the addition of phosphatidyl -ethanolamine, -choline and -serine. After brief treatment with phospholipases A, C and D, enzyme activity is partially lost. After phospholipase A treatment the activity may be completely restored by the addition of phosphatidyl ethanolamine but phosphatidyl-choline and -serine are without effect. After phospholipase C and D treatment, each phospholipid brings about a partial recovery of activity but phosphatidyl ethanolamine is again superior.     

Respiration metabolism in mitochondria of pea seedlings of different age under water deficit and rewatering

The effects of an osmoticum (mannitol) on growth, water content, respiration, and proline content in epicotyls of etiolated two-day-old (S₂) and three-day-old (S₃) pea (Pisum sativum L., cv. Flora-2) seedlings were studied. Water deficit was produced by placing plant roots in 0.6 M mannitol for 2–4 days. Control S₃ epicotyls contained more water than S₂ epicotyls; they were also characterized by more intense mitochondrial activity (in state 3, V ₃) and by the higher value of the respiratory control (RC) due to a fourfold decrease of proportion of weakly phosphorylating cyanide-resistant respiration (CRR). After 2-day-long treatment with the osmoticum, S₂ epicotyls lost more water than S₃; water deficit in them attained 40% (28% in S₃). In S₃ treatment, epicotyls continued to grow at the almost unchanged rate, and the content of proline in them changed insignificantly, whereas in S₂ seedlings epicotyl growth stopped, and proline accumulated in them. In the presence of the osmoticum, V ₃ and RC reduced, but in S₃ treatment they retained higher than in treatment S₂. In mitochondria of S₃ subjected to dehydration, the proportion of the cytochromic pathway of oxidation (V _cyt) remained high, but the absolute values of the respiration rate reduced, whereas in S₂ both these indices reduced and the proportion of CRR increased. After rewatering, indices of mitochondrial activity (the rate of substrate oxidation, RC value) were restored, but in S₃ they were higher. The ratio of V _cyt to CRR in S₃ shifted toward CRR, whereas in S₂, in contrast, it was shifted toward V _cyt. When temperature during seedling growth and experiment performing was declined from 22°C (in control) to 18°C, growth of S₃ epicotyls was retarded to the rate characteristic of S₂ at 22°C. Under the effect of osmoticum, they, like S₂, acquired a capacity to accumulate proline. Nevertheless, mitochondrial activity in such S₃ turned out to be higher than in S₂ grown at 22°C. The results obtained than, as distinct from traditional notes, 3-day-old pea seedlings manifested the higher tolerance to water deficit than 2-day-old seedlings.     

The inhibition of mitochondrial metabolic activity in etiolated pea seedlings under water stress

In the present work, we studied the influence of water (osmotic) stress on mitochondrial metabolic activity in etiolated pea (Pisum sativum L.) seedlings. Three-day-old pea seedlings were subjected to stress by placing their roots in 0.6 M mannitol for 48 h. Epicotyl growth was severely suppressed, and tissue water content was decreased. We revealed the negative influence of the water stress on mitochondrial metabolic activity of seedlings, which effect was retained also after organelle isolation. In particular, in the mitochondria of stressed seedlings, the rate of oxidation of malate and other respiratory substrates (in state 3) was severely decreased, as well as respiratory control ratio. The rate of proline oxidation was reduced most seriously (by 70%). The efficiency of oxidative phosphorylation, according to the ADP/O ratio was not changed or was increased as compared to mitochondria in control plants. Activation of CN-resistant oxidase and other alternative pathways of electron transport in the mitochondrial electron-transport chain in stressed plants were not observed. In the epicotyl tissues under water stress, no MDA was accumulated and proline accumulation was insignificant. The role of mitochondria in adaptation responses of young seedlings is discussed.     

Effect of triethanolamine and silatranes on thermotolerance and accumulation of stress proteins in pea seedlings

A capability of triethanolamine (TEA) and its organosilicon derivatives methyl-, chloromethyl-, and ethoxy silatrane (MS, CMS, and ES, respectively) in low and ultra-low concentrations (from 10^?3 to 10^?13 M) to increase pea (Pisum sativum L.) thermotolerance and the relation of this process with heat-shock protein (HSP) accumulation in the roots were studied. Low and ultra-low concentrations of CMS and MS improved seedling survival under conditions of the heat shock (45°C). This process was not accompanied by stress protein accumulation. ES and CMS affected seedling survival and HSP accumulation differently in dependence on the temperature and preparation concentration.     

Lipoxygenase and hydroperoxide lyase in germinating watermelon seedlings

Lipoxygenase (EC 1.13.1.13) was found in seedlings of Citrullus lanatus (Thunb.) Matsum. and Nakai (watermelon). The enzyme has pH optima of 4.4 and 5.5 and is inhibited by 0.2 mM nordihydroguaiaretic acid. It is present in two functional units with estimated molecular weights of 120,000 and 240,000, respectively.     

增强UV-C辐射作用下小麦和豌豆幼苗光合响应及其相对抗性

以小麦和豌豆为材料,研究了UV-C辐射(波长<280 nm)对叶片光合特性及抗氧化酶活性的影响.结果表明: UV-C辐射增强,可使豌豆叶片光合速率减弱,气孔导度、胞间CO₂浓度、蒸腾速率和羧化效率明显降低,而对小麦叶片上述各项指标的影响则是先增加、后降低;在UV-C辐射下,豌豆的CO₂补偿点逐渐升高,而小麦的CO₂补偿点先降低、后升高.UV-C辐射除了使豌豆的POD活性和小麦的SOD活性逐渐降低外,其他抗氧化酶活性则呈先升高、后降低的变化趋势.小麦对短时间UV-C辐射的抗性比豌豆强,但随着UV-C辐射时间的延长,小麦和豌豆的抗氧化酶活性均降低,光合作用减弱.     

Survey of volatile oxylipins and their biosynthetic precursors in bryophytes

Oxylipins are metabolites which are derived from the oxidative fragmentation of polyunsaturated fatty acids. These metabolites play central roles in plant hormonal regulation and defense. Here we survey the production of volatile oxylipins in bryophytes and report the production of a high structural variety of C5, C6, C8 and C9 volatiles of mosses. In liverworts and hornworts oxylipin production was not as pronounced as in the 23 screened mosses. A biosynthetic investigation revealed that both, C18 and C20 fatty acids serve as precursors for the volatile oxylipins that are mainly produced after mechanical wounding of the green tissue of mosses.     

Cell division and DNA topisomerase I activity in root meristems of pea seedlings during water stress

ABSTRACT

Low water potential, generated by PEG addition to the liquid medium of hydroponically grown pea seedlings, induces a fall in moisture content in the roots, followed by the arrest of elongation. This water stress reduces the mitotic index of root meristems during the treatment and induces the appearance of a peak of mitosis at 12 hours from the beginning of recovery. This peak suggests that during water stress the cell cycle is blocked in G2 or late S phase. In a first attempt to understand the biochemical events leading to cell cycle arrest, we tested the in vitro activity of DNA topoisomerase I extracted from stressed or control root meristems. The activity of this enzyme in extracts from stressed seedlings was lower than in controls, whereas it was higher in extracts from seedlings which had recovered from water stress for a few hours. The highest specific activity was observed with seedlings at 24 hours from the start of recovery. The fact that during stress treatments and recovery there was no variation in the synthesis of a 45 kDa protein, indicated as DNA topoisomerase I, suggested that the activity of this enzyme could be posttranslationally regulated. The hypothesis that variations in the concentration of unknown endogenous regulators of the activity of this enzyme may take place during water loss or uptake in the cytosol of meristematic cells is discussed.     

On ethylene and stem elongation in green pea seedlings

Maximum elongation of excised internodal stem sections of light-grown pea (Pisum sativum L.) seedlings occurred at 10⁻⁵ molar indoleacetic acid (IAA), with submaximal responses occurring at 10⁻⁴ and 10⁻³ molar. Accompanying elongation at concentrations of IAA of 10⁻⁶ to 10⁻³ molar was production of ethylene, with the amount increasing up to 10⁻⁴ molar IAA and then becoming nearly constant. Elongation of light-grown sections was not inhibited by exogenous ethylene up to 10,000 ppm in the presence of 10⁻⁵ molar IAA. Marked (up to 50%) inhibition of elongation of internodal segments in situ was observed after treating whole light-grown seedlings with exogenous ethylene for 20 hours. It is concluded that ethylene is not responsible for the submaximal elongation responses of green pea stem sections at high auxin concentrations, but that IAA per se is accountable.     

Hydroperoxide lyase in rabbit leukocytes: conversion of 15-hydroperoxyeicosatetraenoic acid to 15-keto-pentadeca-5,8,11,13-tetraenoic acid

Incubation of 15-HPETE with rabbit peripheral blood leukocytes resulted in the generation of 8,15-diHETE, 14,15-diHETE, 5,15-diHETE, 15-HETE and a polar metabolite with a retention time on RP-HPLC of 9.5 min, U.V. max at 280 nm. Reduction of this polar metabolite with NaBH4 shifted the U.V. max to 233 nm, suggesting the presence of a conjugated dienone system. Electron impact GC/MS analysis on the polar metabolite revealed a structure of a C-15 short chain aldehyde: 15-keto-pentadeca 5,8,11,13-tetraenoic acid. The formation of this new metabolite is proposed to be catalyzed by the enzyme hydroperoxide lyase. Thus, it is possible that the presence of hydroperoxide lyase activity in leukocytes not only provide a new mechanism for the transformation of hydroperoxides it also may provide a de novo protective effect by controlling the level of intracellular arachidonic acid derived hydroperoxides as well as further prevented their clastogenic action and cellular damage.     

Multiple forms of hydroxycinnamate : CoA ligase in etiolated pea seedlings

A survey of a range of plant tissues showed that the hydroxycinnamate CoA ligase in crude extracts of pea shoots had a high relative activity towards sinapic and other methoxycinnamic acids, together with high activity with p-coumaric acid. The pea enzyme has been resolved by chromatography on DEAE-cellulose into two peaks which differ in their substrate specificity. The form which elutes at relatively low salt concentrations has a ratio activity towards p-coumaric and sinapic acids of about 1.8:1 while the form eluting at higher salt concentrations, although showing very high activity with p-coumaric acid, is inactive towards sinapic acid. The pattern of elution of these forms following gel filtration on Ultragel AcA 34 and Sephadex G100 suggests that these two isoenzymes which differ in ionic properties and substrate specificity can exist in two or three molecular weight forms and there is evidence that these forms are under certain circumstances interconvertible.     

Plastid targeting and transient expression of rat liver ATP: citrate lyase in pea protoplasts

Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide. Received: 14 June 1996 / Revision received: 24 November 1996 / Accepted: 4 January 1997     

Physiological and proteomic alterations in rice (Oryza sativa L.) seedlings under hexavalent chromium stress

Main Conclusion

Rice plants employ two strategies to cope with Cr toxicity: immobilizing Cr ions into cell walls to reduce its translocation and activating antioxidant defense to mitigate Cr-induced oxidative stress. The investigation aimed at understanding the physiological and proteomic responses of rice seedlings to hexavalent chromium (Cr⁶⁺) stress was conducted using two rice genotypes, which differ in Cr tolerance and accumulation. Cr toxicity (200 µM) heavily increased the accumulation of H₂O₂ and \({\text{O}}_{2}^{{ \cdot-}}\) , enhanced lipid peroxidation, decreased cell viability and consequently inhibited rice plant growth. Proteomic analyses suggest that the response of rice proteome to Cr stress is genotype- and Cr dosage-dependent and tissue specific. Sixty-four proteins, which show more than fourfold difference under either two Cr levels, have been successfully identified. They are involved in a range of cellular processes, including cell wall synthesis, energy production, primary metabolism, electron transport and detoxification. Two proteins related to cell wall structure, NAD-dependent epimerase/dehydratase and reversibly glycosylated polypeptide were greatly up-regulated by Cr stress. Their enhancements coupled with callose accumulation by Cr suggest that cell wall is an important barrier for rice plants to resist Cr stress. Some enzymes involved in antioxidant defense, such as ferredoxin-NADP reductase, NADP-isocitrate dehydrogenase, glyoxalase I (Gly I) and glutamine synthetase 1 (GS1) have also been identified in response to Cr stress. However, they were only detected in Cr-tolerant genotype, indicating the genotypic difference in the capacity of activating the defense system to fight against Cr-induced oxidative stress. Overall, two strategies in coping with Cr stress in rice plants can be hypothesized: (i) immobilizing Cr ions into cell walls to reduce its translocation and (ii) activating antioxidant defense to mitigate Cr-induced oxidative stress.     

Changes in the content of endogenous IAA and cytokinins in cotyledons of etiolated pea seedlings during their ontogenesis

The inhibitive growth-correlative effect of cotyledons of pea seedlings decreases during their ontogenesis till the age of 14 days. This decrease is associated with an increase in the level of endogenous cytokinins on the one hand and a decrease of endogenous IAA on the other. This is in harmony with the fact that the correlative-inhibiting effect of pea cotyledon upon the growth of its axillary bud can be weakened by exogenous cytokinin and amplified by exogenous IAA.     

Purification and immunolocalization of an annexin-like protein in pea seedlings

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds ⁴⁵Ca²⁺ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis The authors thank Dennis Brown (Cell Research Institute) for use of this research facilities and Zainab Ilahi and Collin Thomas for valuable technical assistance. Portions of this work were presented at the 1989 and 1990 meetings of the American Society for Cell Biology (Clark et al. 1989, 1990). This work was in part supported by a National Institute of Health Training Grant 1-T32-HD07296-01A3 and by a National Aeronautics and Space Administration grant NAGW 1519.     

Nonprotein amino acids in edible lentil and garden pea seedlings

Summary. Commercial edible seedlings of garden pea (Pisum sativum L.) and lentil (Lens culinaris L.) contain high concentration of nonprotein amino acids and trigonelline. Both seedlings grown in the laboratory or purchased in a supermarket were studied by HPLC. Samples from both origins contained trigonelline, α-aminoadipic acid, homoserine, β-(isoxazolin-5-on-2-yl)-alanine (BIA), and γ-glutamyl-BIA. Garden pea seedlings also contained a uracil-alanine derivative (isowillardiine) in substantial amount. Some of these compounds such as BIA and α-aminoadipic acid have neurotoxic activity. Received December 17, 1999 Accepted February 15, 2000