Nitrate uptake and nitrite release by tomato roots in response to anoxia

Excised root systems of tomato plants (early fruiting stage, 2nd flush) were subjected to a gradual transition from normoxia to anoxia by seating the hydroponic root medium while aeration was stopped. Oxygen level in the medium and respiration rate decreased and reached very low values after 12 h of treatment, indicating that the tissues were anoxic thereafter. Nitrate loss from the nutrient solution was strongly stimulated by anoxia (after 26 h) concomitantly with a release of nitrite starting only after 16 h of treatment. This effect was not observed in the absence of roots or in the presence of tungstate, but occurred with whole plants or with sterile in vitro cultured root tissues. These results indicate that biochemical processes in the root involve nitrate reductase. NR activity assayed in tomato roots increased during anoxia. This phenomenon appeared in intact plants and in root tissues of detopped plants. The stimulating effect of oxygen deprivation on nitrate uptake was specific; anoxia simultaneously entailed a release of orthophosphate, sulfate, and potassium by the roots. Anoxia enhanced nitrate reduction by root tissues, and nitrite ions were released into xylem sap and into medium culture. In terms of the overall balance, the amount of nitrite recovered represented only half of the amount of nitrate utilized. Nitrite reduction into nitric oxide and perhaps into nitrogen gas could account for this discrepancy. These results appear to be the first report of an increase in nitrate uptake by plant roots under anoxia of tomato at the early fruiting stage, and the rates of nitrite release in nutrient medium by the asphyxiated roots are the fastest yet reported.     

Involvement of nitrate reduction in the tolerance of tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.) plants to prolonged root hypoxia

The putative role of nitrate and nitrate reductase in the tolerance to prolonged hypoxia was investigated in tomato plants. Nitrogen nutrition has been modified either by deprivation of nitrate or by addition of tungstate—an inhibitor of nitrate reductase (NR)—in the culture medium. In the absence of nitrate as well as in the presence of tungstate, plant growth was significantly disturbed. In the presence of nitrate, the growth of hypoxic plants maintained, nitrate absorption and NR activity increased and a significant release of nitrite into the medium was observed. This mechanism of nitrate reduction, called nitrate respiration, could be an alternative pathway to oxygen-dependent respiration during root hypoxia and a transient adaptation of tomato roots to hypoxic conditions.     

Nitrate reduction and compartmentation in tomato leaves. I. Nitrate accumulation in leaf sections in aqueous and gaseous medium

Nitrate pools in tomato ( Lycopersicon esculentum Mill. cv. Azes) leaf sections were estimated. Nitrite accumulation in aqueous medium was found to be an inadequate estimate of nitrate pools in tomato leaves. The main reason for the cessation of nitrite accumulation was not depletion of nitrate in the metabolic pool but rather a rapid decay of nitrate reductase (NR) activity as measured by nitrite accumulation in vivo and in vitro. Nitrate diffuses out of the tissue into the medium at a rate higher than the accumulation of nitrite in the tissue. Nitrate leakage from the tissue accelerates the loss of NR activity. Nitrite accumulation in leaf sections kept in an anaerobic gaseous atmosphere ceased earlier than in aqueous medium, at a time when NR activity was still relatively high. Measuring nitrite accumulation in gaseous atmosphere is preferable since NR is more stable and movements of nitrate between pools more restricted.     

Selenium as a sulphydrylic group inductor in plants

We investigated the effect of selenium form and dose on the total glutathione and non-protein -SH group contents in the edible spinach (Spinacia oleracea L.) and ground tomato (Lycopersicon esculentum Mill.) plants. Our experiments were carried out in a hydroponic culture. Selenium was added to the culture medium in its selenite (Na2SeO3 x 5H2O) and selenate (Na2SeO4) forms. Regardless of the selenium form, we observed an increase in the non-protein thiol content. The non-protein -SH group content depended on the form and dose of selenium as well as on the organ and plant species. Regardless of the selenium form, a higher content of non-protein -SH groups were found in the spinach biomass than in the tomato biomass. Selenite contributed to a larger accumulation of non-protein -SH groups in the roots, whereas selenate contributed to their accumulation in the shoots     

Relationships between nitrite reductase activity and genotype-dependent callus growth in rice cell cultures

The nitrite ion content and activity of nitrate reductase and nitrite reductase were examined in scutellum-derived calluses of rice varieties using a modified R2 medium (medium A) and a medium derived from the modified R2 medium (medium B). In medium A, marked differences were observed in callus growth between the varieties. The calluses of the poor-growth varieties accumulated significantly more nitrite ions during the culture period than did the good-growth varieties. Callus growth rate was negatively correlated with the nitrite ion content, indicating that the calluses of the poor-growth varieties were injured by toxic nitrite ions, which lead to browning and inhibited growth. The calluses of the poor-growth varieties had significantly lower levels of nitrite reductase activity than good-growth varieties. On the other hand, no between-group differences were observed in the nitrate reductase activity. These results indicate that the higher nitrite ion levels observed in the poor-growth varieties resulted from a lower ability to reduce nitrite and that nitrite reductase activity is one of the physiological factors that correlates with differences between varieties in rice cell cultures. In medium B, the calluses of the poor-growth varieties grew as well as the good-growth varieties, but also had significantly lower levels of nitrite reductase. Nitrate reductase activity was repressed in the calluses of both varieties in medium B compared to culture in medium A. The results suggest that repressed nitrate reductase activity causes the calluses of poor-growth varieties to accumulate only trace amounts of nitrite ions despite lower nitrite reductase activity and as a result, callus growth improved in medium B. Received: 28 July 1998 / Revision received: 14 October 1998 / Accepted: 27 October 1998     

A study of the nitrate and nitrite discharge from the mutant cells of Neurospora crassa lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

Ion uptake and structural modifications induced by nitrogen source in tomato (Solanum lycopersicum Mill. Cv. Ibiza F1)

Interactions between NO(3)(-) and NO(2)(-) were studied at the level of root uptake, ion translocation (NO(3)(-), NO(2)(-), K(+)), ion xylem exudates composition and inorganic cation contents (K(+), Ca(2+), Mg(2+)) using tomato seedling (Solanum lycopersicum Mill cv. Ibiza F1). Nitrite was supplied in the medium as KNO(2) (0, 0.25, 2.5, 5 and 10?mM). Plants cultivated on the same doses of KNO(3) served as control. The experimental system allowed direct measurements of net NO(3)(-) and NO(2)(-) uptake. Our results showed that NO(3)(-) uptake and translocation were stimulated by external supply of K(+), while they were hardly decreased by NO(2)(-) supply. Contents of K(+) and Mg(2+) were negatively affected in all tomato tissues by increasing nitrite concentration in the medium. Highest dose of NO(2)(-) decreased Ca(2+) accumulation in shoots and conversely increased that in the roots. Histological study at the stem level revealed that nitrite (10?mM) induced a restriction of the tissue territories as well as less developed regions and some conductor tissues disorganization in this organ structure. The overall results suggest that nitrite exposure delayed growth and injured cell structure and overall nutrient uptake.     

Factors effecting the toxicity of nitrite nitrogen to tomatoes

Experiments were done to study the effects of nitrite nitrogen on nutrient absorption and organic acid content of tomatoes (Keystone) grown in sand culture. The effects of root aeration, magnesium and iron supply on the symptoms of nitrite toxicity were also studied. Nutrient solutions were standardised to pH 4.5 and contained from 0–250 ppm nitrite nitrogen. Increasing the concentration of nitrite nitrogen decreased dry matter yields, total acidity, the concentration of nitrogen, phosphorus and potassium in tomato plants, and increased the chlorosis of leaves and the lignification of roots. Shortage of iron, magnesium, and poor root aeration caused toxicity symptoms to appear at a smaller concentration of nitrite nitrogen and increased the severity of the symptoms.     

Transformation of Cucumis melo cv. Hetau with Tomato Antisense ACC Synthase Gene

An efficient in vitro plant regeneration system of Cucumis melo L. cv. Hetau was established. Regenerated plantlets were obtained from cotyledons after preculture, shoot inducing culture and root inducing culture. A high regeneration rate was achieved up to 58%. Cucumis melo was transformed with the antisense tomato ACC synthase gene in binary vector pMQ6 via Agrobacterium tumefaciens mediated gene transfer. Kanamycin resistant plantlets were obtained on MS medium with 6 mg/L zeatin, 50 mg/L kanamycin and 650 mg/L cefotaximine. PCR and molecular hybridization analysis showed that tomato ACC synthase antisense cDNA was integreted into the genome of C. melo.     

Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures

A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv. Seokwang) using Agrobacterium-mediated transformation. Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato. The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene. Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 g l^–1 after 10 d' cultivation.     

Induced androgenesis in tomato (Lycopersicon esculentum Mill). II. Factors affecting induction of androgenesis

The influence on androgenesis of donor plant growth conditions, anther size and developmental stage of the microspore, medium composition and different anther treatments prior to culture was investigated in L. esculentum Mill. cv Roma and its hybrids. Growth conditions of donor plants affected the induction of tomato androgenesis. Anthers isolated from plants grown in the greenhouse during winter at high humidity and in short days possessed higher androgenetic ability than those grown in the field. The physiological state and age of the donor plants also influenced the processes investigated. Regarding the developmental stage of microspores, the period from prophase to telophase II is optimal for tomato anther implantation. More then 20 culture media were tested. Two, based on Murashige and Skoog medium were selected as most favourable for callus induction, organogenesis and regeneration. The effect on callus induction of 2ip in combination with indole-3-acetic acid (IAA) was greater than that of zeatin and IAA. Zeatin promoted entire plant regeneration. A highly significant interaction between genotype and medium was observed. Temperature and gamma ray treatments of anthers enhanced callus production, shoot formation and plant regeneration. Treatments at 4 °C (48 h) and 10 °C (9 days) stimulated these processes. Combined treatment of anthers with 4 Gy and 10 °C for 9 days was the most efficient. Received: 5 September 1997 / Revision recieved: 5 June 1998 / Accepted: 15 June 1998     

钙与植物乙烯反应的关系研究

研究了Ca2 对番茄 (LycopersiconesculentumMillcv.Lichun)黄化幼苗乙烯反应的影响。通过测定不同Ca2 浓度条件下番茄黄化幼苗的“三重反应”、内源乙烯释放量、乙烯受体基因NEVER_RIPE(NR)表达量及胞内CaM含量的变化 ,结果发现 ,随着培养基中Ca2 浓度从 0mmol/L增加到 3.8mmol/L ,番茄黄化幼苗的“三重反应”表型明显增强 ,内源乙烯释放量、NR基因的表达量及胞内CaM的含量都有不同程度的增加 ;当Ca2 浓度由 3.8mmol/L进一步增加到 10mmol/L时 ,番茄黄化幼苗“三重反应”表型受到抑制 ,内源乙烯释放量、NR基因的表达量及胞内CaM的含量都有所下降。因此 ,Ca2 对番茄黄化幼苗“三重反应”的影响与Ca2 调节内源乙烯合成和乙烯受体基因的表达有关 ,而且Ca2 可能是通过CaM含量的变化来调节乙烯作用的     

钙与植物乙烯反应的关系研究

研究了Ca2+ 对番茄(Lycopersicon esculentum Mill cv. Lichun)黄化幼苗乙烯反应的影响.通过测定不同Ca2+ 浓度条件下番茄黄化幼苗的"三重反应"、内源乙烯释放量、乙烯受体基因NEVER-RIPE(NR)表达量及胞内CaM含量的变化,结果发现,随着培养基中Ca2+ 浓度从0 mmol/L增加到3.8 mmol/L,番茄黄化幼苗的"三重反应"表型明显增强,内源乙烯释放量、NR基因的表达量及胞内CaM的含量都有不同程度的增加;当Ca2+ 浓度由3.8 mmol/L进一步增加到10 mmol/L时,番茄黄化幼苗"三重反应"表型受到抑制,内源乙烯释放量、 NR基因的表达量及胞内CaM的含量都有所下降.因此,Ca2+ 对番茄黄化幼苗"三重反应"的影响与Ca2+ 调节内源乙烯合成和乙烯受体基因的表达有关,而且Ca2+ 可能是通过CaM含量的变化来调节乙烯作用的.     

A study of the nitrate and nitrite discharge from the mutant cells of <Emphasis Type="Italic">Neurospora crassa</Emphasis> lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

秸秆浸提液漂浮栽培对不结球白菜产量及相关品质特性的影响

以1/4园试营养液为对照,以腐熟发酵的农业秸秆(苋菜和番茄植株残体)浸提液模拟富含N、P的水体进行不结球白菜(Brassica campestris ssp.chinensis Makino)的漂浮栽培,对茎叶的单株鲜质量和干质量及叶片的VC、可溶性蛋白质、蔗糖、果糖、葡萄糖含量及可溶性糖总含量进行了比较分析。结果表明:在24 d的栽培周期内,随生长时间的延长,处理组和对照组茎叶的单株鲜质量和干质量均呈增加趋势,但从第10天开始,处理组茎叶的单株鲜质量和干质量均显著低于对照组。栽培初期叶片VC含量均最高且处理组叶片VC含量显著低于对照组;从第10天开始,叶片VC含量降低,但处理组叶片VC含量与对照组无显著差异。处理组与对照组叶片可溶性蛋白质含量均呈波动的变化趋势,但总体差异不显著。处理组叶片蔗糖含量总体上与对照组差异不显著,仅在第24天显著低于对照组。处理组叶片的果糖含量均高于对照组,特别是在第24天为对照组的1.81倍,差异显著。叶片葡萄糖含量均呈先升高后降低的趋势,可溶性糖总含量则呈波动的变化趋势,对照组叶片葡萄糖含量和可溶性糖总含量总体上高于处理组,但差异不显著。研究结果显示:用秸秆浸提液漂浮栽培,虽然不结球白菜的生长量(即产量)有所减少,但相关的品质指标变化不大,对果糖合成和积累还有一定的促进作用,因而,此种秸秆浸提液可用于栽培商品不结球白菜。     

Protection of tomato seedlings against infection by Pseudomonas syringae pv. tomato by using the plant growth-promoting bacterium Azospirillum brasilense

Pseudomonas syringae pv. tomato, the causal agent of bacterial speck of tomato, and the plant growth-promoting bacterium Azospirillum brasilense were inoculated onto tomato plants, either alone, as a mixed culture, or consecutively. The population dynamics in the rhizosphere and foliage, the development of bacterial speck disease, and their effects on plant growth were monitored. When inoculated onto separate plants, the A. brasilense population in the rhizosphere of tomato plants was 2 orders of magnitude greater than the population of P. syringae pv. tomato (10(7) versus 10(5) CFU/g [dry weight] of root). Under mist chamber conditions, the leaf population of P. syringae pv. tomato was 1 order of magnitude greater than that of A. brasilense (10(7) versus 10(6) CFU/g [dry weight] of leaf). Inoculation of seeds with a mixed culture of the two bacterial strains resulted in a reduction of the pathogen population in the rhizosphere, an increase in the A. brasilense population, the prevention of bacterial speck disease development, and improved plant growth. Inoculation of leaves with the mixed bacterial culture under mist conditions significantly reduced the P. syringae pv. tomato population and significantly decreased disease severity. Challenge with P. syringae pv. tomato after A. brasilense was established in the leaves further reduced both the population of P. syringae pv. tomato and disease severity and significantly enhanced plant development. Both bacteria maintained a large population in the rhizosphere for 45 days when each was inoculated separately onto tomato seeds (10(5) to 10(6) CFU/g [dry weight] of root). However, P. syringae pv. tomato did not survive in the rhizosphere in the presence of A. brasilense. Foliar inoculation of A. brasilense after P. syringae pv. tomato was established on the leaves did not alleviate bacterial speck disease, and A. brasilense did not survive well in the phyllosphere under these conditions, even in a mist chamber. Several applications of a low concentration of buffered malic acid significantly enhanced the leaf population of A. brasilense (>10(8) CFU/g [dry weight] of leaf), decreased the population of P. syringae pv. tomato to almost undetectable levels, almost eliminated disease development, and improved plant growth to the level of uninoculated healthy control plants. Based on our results, we propose that A. brasilense be used in prevention programs to combat the foliar bacterial speck disease caused by P. syringae pv. tomato.     

Prolonged root hypoxia induces ammonium accumulation and decreases the nutritional quality of tomato fruits

Here we examined the effects of root hypoxia (1-2% oxygen) on the physiology of the plant and on the biochemical composition of fruits in tomato (Solanum lycopersicum cv. Micro-Tom) plants submitted to gradual root hypoxia at first flower anthesis. Root hypoxia enhanced nitrate absorption with a concomitant release of nitrite and ammonium into the medium, a reduction of leaf photosynthetic activity and chlorophyll content, and an acceleration of fruit maturation, but did not affect final fruit size. Quantitative metabolic profiling of mature pericarp extracts by (1)H NMR showed that levels of major metabolites including sugars, organic acids and amino acids were not modified. However, ammonium concentration increased dramatically in fruit flesh, and ascorbate and lycopene concentrations decreased. Our data indicate that the unfavorable effects of root hypoxia on fruit quality cannot be explained by two of the well-known effects of root hypoxia on the plant, namely a decrease in photosynthesis or an excess in ethylene production, but may instead result from disturbances in the supply of either growth regulators or ammonium, by the roots.     

Reversibility of the effects of cadmium on the growth and nitrogen metabolism in the tomato(Lycopersicon esculentum)

In order to better understand the effects of heavy metals on the growth of plants, we decided to perform recovering experiments by following both chemical and physiological parameters in cadmium pre-stressed tomato seedlings after cadmium had been removed from the nutrient solution. The work shows that cadmium suppression results in resumption of growth activity. The biomass of leaves and stems rose steadily. The increase in root biomass exceeded those of leaves and stems. At the same time, nitrate content was increased to reach the level obtained with unstressed controls. In all the organs studied, the activities of the enzymes involved in the anabolic nitrogen primary assimilation pathways (nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) soared after that cadmium had been removed. While NAD(+)-dependent glutamate dehydrogenase (GDH-NAD+) activity also rose progressively during the recovering time, the cognate NADH-dependent glutamate dehydrogenase (GDH-NADH) activity decreased. This result allows us to propose that the ammonia produced by the stress-induced protein catabolism is detoxified and re-assimilated by the GDH-NADH isoenzyme. On the basis of these results, we will discuss the ability of the plant to dilute the effects of pollutants during the recovering period. An important outcome of this work is that a transient contamination of the culture medium by pollutants is not necessarily followed by a significant depreciation in product yield or quality.     

Bacterial siderophores efficiently provide iron to iron-starved tomato plants in hydroponics culture

Iron is one of the essential elements for a proper plant development. Providing plants with an accessible form of iron is crucial when it is scant or unavailable in soils. Chemical chelates are the only current alternative and are highly stable in soils, therefore, posing a threat to drinking water. The aim of this investigation was to quantify siderophores produced by two bacterial strains and to determine if these bacterial siderophores would palliate chlorotic symptoms of iron-starved tomato plants. For this purpose, siderophore production in MM9 medium by two selected bacterial strains was quantified, and the best was used for biological assay. Bacterial culture media free of bacteria (S) and with bacterial cells (BS), both supplemented with Fe were delivered to 12-week-old plants grown under iron starvation in hydroponic conditions; controls with full Hoagland solution, iron-free Hoagland solution and water were also conducted. Treatments were applied twice along the experiment, with a week in between. At harvest, plant yield, chlorophyll content and nutritional status in leaves were measured. Both the bacterial siderophore treatments significantly increased plant yield, chlorophyll and iron content over the positive controls with full Hoagland solution, indicating that siderophores are effective in providing Fe to the plant, either with or without the presence of bacteria. In summary, siderophores from strain Chryseobacterium C138 are effective in supplying Fe to iron-starved tomato plants by the roots, either with or without the presence of bacteria. Based on the amount of siderophores produced, an effective and economically feasible organic Fe chelator could be developed.