Treatment Of Leaf Discs Research Articles

ДИНАМИКА НАКОПЛЕНИЯ САХАРОВ И ФЛАВОНОИДОВ В ЛИСТЬЯХ ГРУШИ ПРИ МОДЕЛИРОВАННОМ ОСМОТИЧЕСКОМ СТРЕССЕ

The purpose of the study is to conduct a comparative assessment of three pear varieties in terms of the content of soluble sugars and flavonoids under simulated osmotic stress. The objects of the study are three varieties of pear Pyrus communis L.: two varieties Williams and Conference of European origin and one domestic variety Flamenco selected by the North Caucasus Federal Scientific Center for Horticulture, Viticulture, and Winemaking. Drought was simulated in two ways. In the first case, the leaves were cut into leaf disks with a diameter of 1 cm, then immersed in a 20 % solution of polyethylene glycol (PEG-6000) for 2 hours at room temperature. Control is buffer solution not containing polyethylene glycol. The second method is to place pear shoots in a solution of 15 % sorbitol for a day at room temperature, where water was the control. Exposure to 15 % sorbitol caused more intense synthesis of primary and secondary metabolites compared to 20 % PEG solution. In July, an increase in the accumulation of sugars compared to the control was noted in the Conference variety (25.6 mg glucose equivalent/g fresh weight), in August this figure sharply increased in the Flamenco and Williams varieties to 24.3 and 25.7 mg equivalent. glucose/g wet weight, respectively. A significant increase in flavonoid content in response to sorbitol stress was detected in August for the Flamenco and Conference varieties, in which the average values changed from 2.1 and 2.5 to 3.6 and 4.2 mg/g fresh weight, respectively. Treatment of leaf disks with PEG did not initiate an increase in the synthesis of primary and secondary metabolites. A more effective way to create osmotic stress in the tissues of pear leaves was treatment with a sorbitol solution.

Suppression of tomato SlGGP aggravates methyl viologen-mediated oxidative stress

Ascorbate (AsA) is an important antioxidant that can scavenge reactive oxygen species to protect plant cells against oxidative stress. Guanosine 5'-diphosphate (GDP)-L-galactose phosphorylase (GGP) is a key enzyme in the AsA biosynthetic pathway. To investigate the functions of GGP in AsA synthesis and oxidative stress tolerance in tomato, antisense lines with a reduced expression of SlGGP were obtained. Photobleaching after treatment of leaf disks with methyl viologen was more severe in transgenic lines compared to wild type (WT) plants. Moreover, compared with the WT plants, the transgenic plants showed a higher content of hydrogen peroxide, superoxide anion, malondialdehyde, as well as ion leakage, but a lower content of AsA and chlorophylls, ascorbate peroxidase activity, net photosynthetic rate, and maximal photochemical efficiency of photosystem II. Results of real-time quantitative polymerase chain reaction show that suppression of the SlGGP gene in the transgenic plants reduced their oxidative stress tolerance.

In vivo inhibition of cysteine proteases provides evidence for the involvement of ‘senescence-associated vacuoles’ in chloroplast protein degradation during dark-induced senescence of tobacco leaves

Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

Methyl jasmonate-induced cell death in grapevine requires both lipoxygenase activity and functional octadecanoid biosynthetic pathway

Grapevine (Vitis vinifera L., cv. Limberger) leaf tissues and suspension-cultured cells were induced to undergo programmed cell death (PCD) by exogenously added methyl jasmonate (MeJA). The elicitor signaling pathway involved in MeJA-induced PCD was further investigated using pharmacological, biochemical and histological approaches. Pharmacological dissection of the early events preceding the execution of MeJA-triggered PCD indicated that this process strongly depends on both, de novo protein and mRNA synthesis. Treatment of leaf discs and cell suspensions with lipase inhibitor Ebelactone B and specific lipoxygenase inhibitor Phenidone blocked MeJA-induced PCD. These results suggest that some chloroplast membrane-derived compound(s) is required for MeJA-induced PCD in grapevine. The progression of MeJAtriggered PCD may be further inhibited by the use of metabolic inhibitors of key enzymes of octadecanoid biosynthesis including AOS, AOC, and OPR indicating that the functional jasmonate biosynthetic pathway is an integral part of the MeJA-induced signal transduction cascade that results in the coordinate expression of events leading to PCD. Finally, the activation of the octadecanoid pathway, as a critical point in MeJA-induced PCD, was independently demonstrated with cellulysin, a macromolecular elicitor acting via octadecanoid signaling. The cellulysin was shown to be a very potent enhancer of MeJA-triggered PCD in grapevine cells.

Involvement of phospholipase D and phosphatidic acid in the light-dependent up-regulation of sorghum leaf phosphoenolpyruvate carboxylase-kinase

The photosynthetic phosphoenolpyruvate carboxylase (C4-PEPC) is regulated by phosphorylation by a phosphoenolpyruvate carboxylase kinase (PEPC-k). In Digitaria sanguinalis mesophyll protoplasts, this light-mediated transduction cascade principally requires a phosphoinositide-specific phospholipase C (PI-PLC) and a Ca2+-dependent step. The present study investigates the cascade components at the higher integrated level of Sorghum bicolor leaf discs and leaves. PEPC-k up-regulation required light and photosynthetic electron transport. However, the PI-PLC inhibitor U-73122 and inhibitors of calcium release from intracellular stores only partially blocked this process. Analysis of [32P]phosphate-labelled phospholipids showed a light-dependent increase in phospholipase D (PLD) activity. Treatment of leaf discs with n-butanol, which decreases the formation of phosphatidic acid (PA) by PLD, led to the partial inhibition of the C4-PEPC phosphorylation, suggesting the participation of PLD/PA in the signalling cascade. PPCK1 gene expression was strictly light-dependent. Addition of neomycin or n-butanol decreased, and a combination of both inhibitors markedly reduced PPCK1 expression and the concomitant rise in PEPC-k activity. The calcium/calmodulin antagonist W7 blocked the light-dependent up-regulation of PEPC-k, pointing to a Ca2+-dependent protein kinase (CDPK) integrating both second messengers, calcium and PA, which were shown to increase the activity of sorghum CDPK.

Mechanism of CATA3 induction by cadmium in sunflower leaves

One of the main antioxidant enzymes, catalase (CAT, EC 1.11.1.6), is capable of catalyzing the dismutation of H 2O 2. This enzyme is involved in signal transduction pathway in plants, controlling the cellular level of this reactive oxygen species. Four different genes, CATA1– CATA4, were identified in Helianthus annuus L. cotyledons. Incubation of sunflower leaf discs with 300 and 500 μM CdCl 2 under light conditions increased CATA3 transcript level. However, it was not induced by Cd 2+ in etiolated plants. This Cd 2+-induced increase was reverted by adding 10 mM ascorbate. Treatments with 0.4 and 10 μM rose bengal (a generator of 1O 2) did not activate CATA3, but 10 μM methyl viologen (an enhancer of O 2 − production) and 10 mM H 2O 2 increased its expression. In isolated chloroplasts, Cd 2+ and methyl viologen produced oxidation of the probe 2′,7′-dichlorofluorescein diacetate indicating ROS formation. Besides, Cd 2+ treatment of leaf discs under light decreased CAT activity and increased carbonyl groups content, thus suggesting that enzyme inactivation could be due – in part – to a protein oxidation. These results indicate that light is involved in Cd 2+-induced CATA3 enhancement, which leads to the synthesis of CAT isoforms less sensible to oxidation, and that chloroplast might be the main source of ROS responsible for this process.

Sugar-Induced Increase of Calcium-Dependent Protein Kinases Associated with the Plasma Membrane in Leaf Tissues of Tobacco.

The sugar-inducible expression of genes for sporamin and [beta]-amylase in leaf explants of sweet potato (Ipomoea batatas) and that of a [beta]-glucuronidase-fusion gene, with the promoter of the gene for [beta]-amylase in leaves of tobacco (Nicotiana tabacum), requires Ca2+ signaling (M. Ohto, K. Hayashi, M. Isobe, K. Nakamura [1995] Plant J 7: 297-307), and it was inhibited by staurosporin and K252a, inhibitors of protein kinases. Autophosphorylation activities of several potential protein kinases in leaves of tobacco were significantly higher in younger leaves than in mature leaves. However, the autophosphorylation activities of these proteins in mature leaves, especially those of the major autophosphorylatable proteins with apparent molecular masses of 56 and 54 kD, increased upon treatment of leaf discs with a 0.3 M solution of sucrose, glucose, or fructose, did not increase with sorbitol or mannitol treatments, and the increase by sucrose was inhibited by cycloheximide. Autophosphorylation of the 56- and 54-kD protein in vitro was dependent on Ca2+ and inhibited by staurosporine, K-252a, and by W-7. These results suggest that they belong to the family of calcium-dependent protein kinases. They were concentrated in the plasma membrane fraction and were released from membrane vesicles by high salt or with sodium carbonate. The possible functions of these sugar-inducible calcium-dependent protein kinases associated with the plasma membrane are discussed.

Treatment of Agrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco.

We have studied the effect of the demethylating agent azacytidine (azaC) on expression of a beta-glucuronidase (GUS) gene transferred to tobacco leaf disks by Agrobacterium-mediated transformation. In a system where no selection was performed, where shoot formation was partially repressed, and where Agrobacterium does not express the GUS gene, we were able to follow the early events of transient and stable expression. Two days after inoculation, 8% of the cells expressed GUS but this proportion rapidly decreased to near zero in the following week. Treatment of leaf disks with azaC just after transformation retarded this inactivation to some extent, while treatment of Agrobacterium prior to transformation increased the frequency of transient expression. Three weeks after inoculation the number of GUS-expressing cells increased 4- to 6-fold in the leaf disks treated with azaC and in the leaf disks transformed with azaC-treated bacteria, while the control remained low. These data suggest that DNA methylation is involved in transgene inactivation and that a large number of silent but potentially active transgenes become integrated.

Effect of Exogenous ATP on Nitrate Assimilation in Leaf Discs of Crop Plants

Summary Exogenously supplied (U-3H) ATP is shown to be absorbed by wheat leaf discs. Increase in the endogenous ATP concentration significantly stimulated nitrate assimilation in the dark by leaf discs of a number of crop plants. Treatment of leaf discs with DNP diminished the endogenous glucose-6-phosphate concentration and hence promoted nitrite accumulation under aerobic conditions. Role of ATP in nitrate assimilation is discussed.

HERBICIDAL ACTION OF IODIDE: EFFECT ON CHLOROPHYLL CONTENT AND PHOTOSYNTHESIS IN DWARF BEAN PHASEOLVS VULGARIS

Summary. The toxicity of potassium iodide towards bean plants (Phaseolus vulgaris) is greater in the light than in the dark. Treatment of leaf discs in the dark or the light reduces photosynthetic activity to the same extent, but the loss of chlorophyll is less pronounced in the dark. Iodine behaves similarly but is effective at lower concentrations. Iodide does not inhibit chlorophyll synthesis. Iodine is some 105 times as effective as iodide in inhibition of the Hill reaction in isolated chloroplasts. These results are consistent with the hypothesis that iodine toxicity is a result of intracellular oxidation to iodine. Action herbicide de l'iodure de potassium. Effet sur la teneur en chlorophylle et la photosynthèse sur le haricot nain (Phascolus vulgaris)