Comparative effects of ethylene and cyanide on respiration, polysome prevalence, and gene expression in carrot roots

Treatment of carrot roots (Daucus carota L.) with 10 microliters per liter ethylene in O₂ evokes a three- to four-fold increase in polysome prevalence and associated poly(A)⁺ RNA. The increase in polysome prevalence is attended by a similar change in CO₂ evolution. The increase in polysomal poly(A)⁺ mRNA constitutes primarily a generic increase in constitutive mRNAs as assayed by in vitro translation. However, changes in the relative abundance of several in vitro translatable ethylene specific mRNAs do occur.

Cyanide, at concentrations which inhibit cytochrome oxidase, initiates a respiratory rise very similar in kinetics and magnitude to that evoked by ethylene. Moreover, the combined treatment with cyanide and ethylene evokes a respiratory response resembling that caused by ethylene or cyanide alone. Nevertheless, cyanide, in the presence of ethylene, significantly inhibits the increase in polysome prevalence and new gene expression associated with ethylene treatment of carrot roots. Separation of in vitro translation products by one-dimensional and two-dimensional gel electrophoresis shows that several new in vitro translation products appear in cyanide-treated carrots different from those evoked by ethylene. Engagement of the less energy efficient alternative electron transport path by cyanide may be responsible for inhibition of the normal ethylene associated increase in polysome prevalence and new gene expression. The implications of these results on regulation of respiratory metabolism are discussed and compared with the results for similar experiments with avocado fruit (Tucker and Laties 1984 Plant Physiol 74: 307-315) in which cyanide does not inhibit an ethylene educed increase in polysome prevalence and change in gene expression.

     

Effects of rapid changes in oxygen concentration on the respiration of carrot roots

Rates of CO₂ production and O₂ consumption from aged disks of carrot ( Daucus carota L.) root tissues were measured for 4 h after they were transferred from 21% to 0, 1, 2, 4 or 8% O₂ in gas mixtures. A transient peak in the rate of CO₂ production started 5 to 7 min after transfer to 2% or lower O₂ mixtures and peaked at 50 min. After the peaks in CO₂ production from the 0, 1 and 2% O₂ treatments and after the stable production from the 4 and 8% O₂ treatments, the rate of CO₂ production from all low O₂ treatments started to decline at 50 min, reaching stable rates by 160 to 240 min. Concentrations of lactate and ethanol that were significantly higher than the 21% O₂ controls had started to accumulate in disks between 10 and 50 min after exposure to atmospheres containing 2% or less O₂. Production of CO₂ started to increase 5 to 7 min after transfer to 0, 1 and 2% O₂, while the initial decline and then rise in pH and the accumulation of ethanol did not occur until 30 min after the change in atmosphere. Ethanol accumulation paralleled the increase in pH; first at 0.4 μmol g⁻¹ h⁻¹ from 30 to 60 min as the pH shifted from 5.97 to 6.11, and then at 0.08 μmol g⁻¹ h⁻¹ from 60 to 100 min as the pH stablized around 6.12. The peak at 50 min in CO₂ production roughly coincided with the shift from the rapid to the slow change in pH and ethanol accumulation.     

Phosphofructokinase of carrot roots

Phosphofructokinase was partially purified from carrot root extracts. Monovalent cations stimulated carrot phosphofructokinase activity. The enzyme was strongly inhibited by P-enolpyruvate and this inhibition was relieved by NACl or KCl. Pi inhibited the enzyme at pH 7.9 but was stimulatory at pH 6.6.     

Differential gene expression of rice roots inoculated with the diazotroph Herbaspirillum seropedicae

Background and aims

Rice (Oryza sativa L.) is the primary source of carbohydrate for the majority of the World's population. Herbaspirillum seropedicae is a diazotroph that lives within and on the surface of rice roots. It can promote the growth of rice, partly by supplying it with fixed nitrogen.

Methods

To better understand the rice–H. seropedicae interaction, cDNA libraries from rice roots either inoculated (RRCH) or uninoculated (RRSH) with the diazotroph were obtained and analysed.

Results

Potential differentially expressed genes identified from the libraries encoded a metallothionein-like protein type 1, a NOD26-like membrane integral protein ZmNIP2-1, a thionin family protein, an oryzain gamma chain precursor, stress-associated protein 1 (OsISAP1), probenazole-inducible protein PBZ1 and auxin- and ethylene-responsive genes. Differential expression was analysed by qRT-PCR for some of these genes and confirmed in most cases. The expression of stress- and defence-related genes coding for thionins, PBZ1 and OsISAP1 was repressed, while expression of a metallothionein gene was induced by inoculation with H. seropedicae. In contrast, expression of auxin-responsive genes was repressed, while expression of ethylene genes was either repressed or induced. The possible involvement of these and other genes in plant-bacterial interactions is discussed.

Conclusions

The decrease in expression of the defence-related proteins PBZ1 and thionins in the rice–H. seropedicae association, suggests that the bacteria modulate plant defence responses during colonisation. The expression of genes responsive to auxin and ethylene also appears to be regulated by the bacteria.     

Differential control of cellular gene expression by diffusible and non-diffusible EGF

Cell gene expression is affected by both the kind and mode of growth factor stimulation (diffusive vs. non-diffusive). Epidermal growth factor (EGF) was pattern-immobilized on a polystyrene plate. Although the growth of the rat phaeochromocytoma cell line PC12 is stimulated by diffusible EGF, and differentiation is stimulated by diffusible nerve growth factor (NGF), immobilized (non-diffusible) EGF stimulated PC12 differentiation. The immobilized EGF caused a long-lasting stimulation of the intracellular signal protein mitogen-associated protein MAP kinase (MAPK, also known as ERK) and p38 (a subfamily of the MAPK superfamily) in cells, as did diffusible NGF. The switching between growth stimulation and differentiation is considered to be due to the duration of the stimulus. The function of the biosignal conjugate was regulated using conjugation methodology.     

Tissue-specific accumulation of carotenoids in carrot roots

Raman spectroscopy can be used for sensitive detection of carotenoids in living tissue and Raman mapping provides further information about their spatial distribution in the measured plant sample. In this work, the relative content and distribution of the main carrot (Daucus carota L.) root carotenoids, α-, β-carotene, lutein and lycopene were assessed using near-infrared Fourier transform Raman spectroscopy. The pigments were measured simultaneously in situ in root sections without any preliminary sample preparation. The Raman spectra obtained from carrots of different origin and root colour had intensive bands of carotenoids that could be assigned to β-carotene (1,520 cm⁻¹), lycopene (1,510 cm⁻¹) and α-carotene/lutein (1,527 cm⁻¹). The Raman mapping technique revealed detailed information regarding the relative content and distribution of these carotenoids. The level of β-carotene was heterogeneous across root sections of orange, yellow, red and purple roots, and in the secondary phloem increased gradually from periderm towards the core, but declined fast in cells close to the vascular cambium. α-carotene/lutein were deposited in younger cells with a higher rate than β-carotene while lycopene in red carrots accumulated throughout the whole secondary phloem at the same level. The results indicate developmental regulation of carotenoid genes in carrot root and that Raman spectroscopy can supply essential information on carotenogenesis useful for molecular investigations on gene expression and regulation.     

Effectiveness of some substances in the control of carrot and parsley roots against fungal diseases

Field experiments were carried out in the years 2005 and 2006 on carrot cv. 'Koral' and 'Perfekcja', and parsley cv. 'Berlinska' and 'Cukrowa'. Effectiveness of substances: Biochikol 020 PC (biologically active substances BAS--chitosan 20 g/dm3), Bioczos BR (extract of garlic 10 g/1 brick) and Biosept 33 SL (extract of grapefruit 33%) on seedling roots of carrot and parsley was studied. As the standard fungicide Zaprawa Funaben T (carbendazim 20% + tiuram 45%) was used. Roots of carrot and parsley were treated one of tested substances spring immediately before planting seedling roots. During vegetation period the growth of seedling shoots and setting of seeds, and their infestation by fungal and bacterial pathogens was noticed. Among substances used for spring dressing of carrot and also parsley seedling roots, the best efficacy exhibited Zaprawa Funaben T in both years of observation. The highest yield of carrot seeds had combination roots cv. 'Koral' and parsley seeds roots cvs 'Berlińska' and 'Cukrowa' dressed Zaprawa Funaben T. Effectiveness of biopreparates Biochikol and Biosept was lower in comparison with the standard fungicide, but their protective effect was significantly higher than in control. Bioczos had the lowest control efficacy.     

Alcohol dehydrogenase gene expression in callus and somatic embryo of carrot

A carrot mRNA of about 1700 nucleotides hybridized specifically to a cloned maize alcohol dehydrogenase (Adh) gene. Hybridization of mRNAs from callus and somatic embryo to the maize probe suggests that Adh expression in carrot is similar in these two cell types.     

Regulation of salt respiration in carrot root slices

In slices of carrot-phloem parenchyma washed for 7 days in water at 20 C, 50 mm KCl stimulates respiration by up to 100% of the ground respiration within 4 minutes of application. The data presented imply that ADP liberated in the cytoplasm as a consequence of KCl accumulation first stimulates a regulator reaction requiring ADP (phosphoglycerate kinase). Thereafter, the point of control alternates between this reaction and the phosphofructokinase reaction, forming a sequence of enzyme stimulations which continue after the new steady state of increased respiration is established. KCl induces a similar sequence in slices washed for 3 days, but it is completed within 3 minutes, and metabolite oscillations are not so marked. In slices washed for 2 days, KCl stimulates respiration by less than 10%, and the sequence of regulator reactions does not occur. Phosphoglycerate kinase is the only enzyme stimulated within 3 minutes of applying KCl to these slices. Contrary to previous reports, KCl frequently stimulates the respiration of freshly prepared slices by 10 to 30%.     

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic--semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.     

基因差异性表达与衰老

微细胞融合实验表明：人的第１、４、６、７号染色体以及Ｘ染色体上存在衰老基因;基因差异性表达研究方法显示：衰老细胞具有特异性表达或高表达的基因。提示细胞衰老是个主动过程,可能与包括衰老基因在内一系列基因激活有关。     

Biosynthesis of SOD by carrot hairy roots

Summary Superoxide dismutase(SOD) was produced by carrot hairy roots. After increasing in the early exponential growth phase, SOD biosynthesis decreased as the growth rate of hairy roots declined. High sucrose concentration stimulated the SOD biosynthesis, while inhibiting the growth of hairy roots. Potential inducers were tested for the enhanced production of SOD. The addition of 0.3 mM CuSO₄ to the broth showed about 6 fold increase in SOD biosynthesis compared to the results obtained with basic MS medium. Since CuSO₄ in the broth inhibited the growth of hairy roots, the addition of CuSO₄ to the broth at the late exponential growth phase led to a 12 times higher intracellular level of SOD compared to that in basic MS medium.