Determination of leghemoglobin components and xylem sap composition by capillary electrophoresis in hypernodulation soybean mutants cultivated in the field

The hypernodulation soybean mutant lines (NOD1-3, NOD2-4, NOD3-7) and their parent Williams, and the mutant En6500 and its parent Enrei were cultivated in a sandy dune field in Niigata, and the nodules and root bleeding xylem sap were sampled at 50, 70, 90, and 120 d after planting (DAP). The nodule size distribution patterns and concentration of leghemoglobin components were determined. The number of nodules of the hypernodulation mutant lines was about two to three times higher than that of the parent lines irrespective of the sampling date. At 50 DAP the nodule size was relatively smaller in the hypernodulation mutant lines, and the total dry weight of the nodules was almost the same in the mutant lines and their parents. At 70 DAP and 90 DAP, the size distribution of the hypernodulation mutant nodules became .almost the same as that of the parent lines, and both the number and total dry weight of the nodules were higher than those of the parent lines. The concentration of four Lb components was separately measured by capillary electrophoresis. The concentration of the Lb components in the hypernodulation mutant lines tended to be lower than in the parents, but the component ratios were not different between the hypernodulation mutants and their parents. Under field conditions, plant growth and nodulation characteristics were more similar between mutants and parents than in the hydroponic culture reported previously, although the mutants did exhibit hypernodulation traits. These findings suggest that the decrease in the Lb concentration and the different Lb components ratios in the mutants may be caused by secondary effects of excess nodulation, such as photosynthate deficiency, rather than by a genetic defect in mutation. The concentration of major nitrogenous compounds (allantoic acid, allantoin, asparagine, aspartic acid, and nitrate) in the xylem sap was also measured by capillary electrophoresis. The concentration of ureides and nitrate in xylem sap decreased with the plant age, but the asparagine concentration increased during the same period. The concentrations of ureides and asparagine were higher, and the nitrate concentration was lower in the mutant lines than in their parents, possibly due to the higher dependence on N₂ fixation than N0₃ ^- utilization. In the xylem sap, nitrate was the major inorganic anion followed by phosphate, sulfate, and chloride, and potassium was the major cation followed by calcium or magnesium and sodium.     

Photoassimilate partitioning in hypernodulation mutant of soybean (Glycine max (L.) Merr.) NOD1-3 and its parent williams in relation to nitrate inhibition of nodule growth

Nodule growth of a hypernodulating soybean (Glycine max (L.) Merr.) mutant line NOD1-3 was compared to that of its wild-type parent cv. Williams from 14 to 18 days after planting (DAP) in the absence of nitrate treatment (hereafter referred to as “0 mM treatment”) or with 5 mM nitrate treatment. The growth rate determined by increase in the diameter of the nodules was relatively lower in the mutant NOD1-3 than that of the parent Williams under nitrogen-free conditions (0 mM nitrate). The inhibition of nodule growth by 5 mM nitrate started at 1 d after the onset of the nitrate treatment in Williams, while the inhibition did not occur before the application of the nitrate treatment for 2 d in NOD1-3. The nodule growth was completely inhibited after 2 d in Williams and after 3 d in NOD1-3 during the 5 mM nitrate treatment period. After 4 d of 5 mM nitrate treatment, the nodule dry weight decreased by 22% in NOD1-3 and by 58% in Williams, respectively. The treatment with 5 mM nitrate decreased the acetylene reduction activity (ARA) in NOD1-3 by 60% per plant and by 50% per nodule g DW and these parameters were less sensitive to the treatment than those in Williams in which the inhibition rate was 90% per plant and 80% per nodule g DW. These results indicate that NOD1-3 is partially nitrate-tolerant in terms of individual nodule growth as well as total nodule dry weight and Nz fixation activity. A whole shoot of Williams and NOD1-3 plants was exposed to ¹⁴CO₂ for 120 min followed by 0 or 5 mM nitrate treatment for 2 d, and the partitioning of the photoassimilates among the organs was analyzed. Under 0 mM nitrate treatment, the percentages of the distribution of ¹⁴C radioactivity between the nodules and roots were 63 and 37% in Williams and 89 and 11% in NOD1-3. Under the 5 mM nitrate conditions, the percentages of the distribution of ¹⁴C between the nodules and roots changed to 14 and 86% in Williams and 39 and 61% in NOD1-3, respectively. These results indicated that the hypernodulating mutant NOD1-3 supplied a larger amount of photoassimilates to the nodules than to the roots under nitrogen-free conditions, and that the nitrate depression of photoassimilate transport to the nodules was less sensitive than that of the parent line.     

Evidence that a shoot-derived substance is involved in regulation of the super-nodulation trait in soybean

Results of grafting experiments between super-nodulation (or hyper-nodulation) mutants of soybean and their parents reconfirmed that super nodulation is a shoot-controlled phenomenon, suggesting that a systemic regulatory mechanism acts in soybean plants and a specific nodulation-controlling substance (SNS) is synthesized in the shoot and transported to the roots. To search for the SNS involved in the super-nodulation trait of NOD1-3, a mutant of soybean ( Glycine max [L.] Merr. cv. Williams), we adopted a bioassay system using plantlets derived from the first trifoliate leaf of the seedlings; this system enabled us to introduce liquid substances continuously into leaves and to assess their effect on root nodulation. Following the application of leaf extract from Williams82 plants lacking visible root nodules, formation of root nodule meristems in NOD1-3 plantlets was repressed on the sixth day after rhizobial inoculation and the number of visible nodules on the eighth day declined to the same level as that in the Williams82 plantlets. Application of NOD1-3 leaf extract resulted in no significant change in the nodulation of both NOD1-3 and Williams82 plantlets. These results suggested that the SNS is a downregulator of nodulation and is responsible for the wild-type (Williams82) phenotype, and that the super-nodulation phenomenon is caused by a paucity of the SNS. The intensity of the repressive effect of the Williams82 leaf extract was not changed by nodulation of the source plants, thus we conclude that visible nodule formation is not required to induce production of the SNS.     

Effect of exogenous salicylic acid supply on nodule formation of hypernodulating mutant and wild type of soybean

Soybean plant is characterized by a systemic autoregulatory control system of nodulation (autoregulation) by initial infection with rhizobia, and plants commonly display a systemic acquired resistance (SAR) to pathogenic microbe infection related to salicylic acid (SA) signal transmission. We investigated the effect of exogenous SA supply on soybean nodulation to determine whether SA affects the autoregulation of nodulation. Seedlings of the hypernodulating mutants NOD1-3, NOD2-4 and their parent cv. Williams were treated or not treated (control) with a 100 μmS-SA solution at 5 d before the inoculation of Bradyrhizobium japonicum strain USDA110. The nodule dry weight and the number of nodules of the wild type soybean Williams exhibiting autoregulation drastically decreased by the addition of 100 μm SA. The decrease in the nodule number was not caused by the reduction of the rhizobium number in the medium. Salicylic acid inhibited only early nodule formation and did not affect the growth of formed nodules. The inhibitory effect of SA on the nodulation of NOD1-3 and NOD2-4 was significantly less pronounced than that in Williams. These results indicate that SA is directly involved in signal transmission in the autoregulation, and that SA or the SAR induced by SA stimulates the autoregulation of nodulation in soybean.     

Influence of Phosphate Dissolving Bacteria on the Efficiency of Superphosphate in a Calcareous Soil Cultivated with Vicia faba

In a pot experiment with Vicia faba grown in a calcareous soil and fertilized with three rates on superphosphate, inoculation with phosphate dissolving bacteria (PDB) increased P-uptake and dry weight. The effect of inoculation exceeded that of the fertilization with half of the usual rate. Dry weight and P-uptake of plants grown in inoculated soils receiving one half of the usual rate of superphosphate were higher than those for plants grown in pots receiving the usual rate of superphosphate in the absence of the inoculation. The usual rate of superphosphate in the presence of inoculation doubled both P-uptake and dry weight of Vicia faba. However, one half of the usual rate of superphosphate in the presence of inoculation with PDB increased P-uptake and dry weight of faba plants by one half more than the control.     

Effectiveness of foreign bradyrhizobia strains in enhancing nodulation, dry matter and seed yield of soybean (Glycine max L.) cultivars in Nigeria

 Field experiments were conducted to investigate the performance of three soybean cultivars with five foreign bradyrhizobia strains in different regions. The experiments at the two sites were designed with soybean (Glycine max L.) cultivars as the main factor and bradyrhizobia strains (USDA 136, TAL 122, USDA 6, TAL 377 and TAL 102) as the sub-factor. The experiments were arranged in randomised complete block design with four replications. Results show that nodule number, nodule dry weight and shoot dry weight, total N and seed yield were significantly increased when soybean cultivars were inoculated with foreign bradyrhizobia in two locations in the south east of Nigeria. At 63 days after planting the percentage increase in nodule number and dry weight after inoculation of soybean cultivars with bradyrhizobia strains ranged from 71 to 486% and from 0 to 200%, respectively. The percentage increase in shoot dry matter, %N and total N after bradyrhizobia inoculation ranged between 2–130%, 18–62% and 35–191%, respectively at Awka, and at the Igbariam site the percentage increase in shoot dry weight, %N and total N ranged between 3–76%, 0–43% and 19–125%, respectively. Seed yields after bradyrhizobia inoculation of soybean cultivar TGX 1485–1D at Igbariam ranged between 1.20 and 2.18 t ha^–1 against the uninoculated plants, which had seed yields of 1.05 t ha^–1. The poorest yield response after inoculation with bradyrhizobia strains was observed in soybean cultivar M-351, with a seed yield ranging from 0.60 to 0.98 t ha^–1. The fact that foreign bradyrhizobia strains were more effective than the indigenous strains for all the parameters studied suggests that there is a need to use bradyrhizobia inoculants for increased soybean production in Nigeria. The variations in the strain performance with the different soybean cultivars at the two sites, emphasises the need for careful Bradyrhizobium spp. strain selection. The fact that inoculation response was cultivar- and site-specific suggests that strategies for improving inoculation response in soybean cultivars should also consider the soil environment where the soybean is to be produced. Received: 25 May 1999     

Effects of co-inoculation of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 on plant growth,nodulation, nitrogen fixation,nutrient uptake,and yield of soybean in a field condition

ABSTRACT

Co-inoculation of nitrogen-fixing bacteria with plant growth-promoting bacteria has become more popular than single inoculation of rhizobia or plant-growth-promoting bacteria because of the synergy of these bacteria in increasing soybean yield and nitrogen fixation. This study was conducted to investigate the effects of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 co-inoculation on plant growth, nodulation, nitrogen fixation, nutrient uptake, and seed yield of the ‘Yezin-6’ soybean cultivar. Nitrogen fixation was measured using the acetylene reduction assay and ureide methods. Uptake of major nutrients [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)] was also measured. This study showed that single inoculation of SAY3-7 significantly increased shoot biomass; nodulation; Relative Ureide Index (RUI %), percent nitrogen derived from N fixation (% Ndfa); N, P, K, Ca, and Mg uptakes; during the later growth stages (R3.5 and R5.5), compared with control. These observations indicate that SAY3-7 is an effective N-fixing bacterium for the plant growth, nodulation, and nitrogen fixation with an ability to compete with native bradyrhizobia. Co-inoculation of SAY3-7 and P4 significantly improved nodule number; nodule dry weight; shoot and root biomass; N fixation; N, P, K, Ca, and Mg uptake; at various growth stages and seed yield in ‘Yezin-6’ soybean cultivar compared with the control, but not the single inoculation treatments. Significant differences in plant growth, nodulation, N fixation, nutrient uptake, and yield between co-inoculation and control, not between single inoculation and control, suggest that there is a synergetic effect due to co-inoculation of SAY3-7 and P4. Therefore, we conclude that Myanmar Bradyrhizobium strain SAY3-7 and P4 will be useful as effective inoculants in biofertilizer production in the future.     

RESPONSES OF “NEWHALL” ORANGE TREES TO IRON DEFICIENCY IN HYDROPONICS: EFFECTS ON LEAF CHLOROPHYLL,PHOTOSYNTHETIC EFFICIENCY,AND ROOT FERRIC CHELATE REDUCTASE ACTIVITY

Orange (Citrus sinensis L. Osb. cv. ‘Newhall’) plants grafted on Citrange troyer rootstock were grown in nutrient solution with 0, 5, 10, or 20 μM iron (Fe), with and without calcium carbonate. Calcium carbonate was added in order to mimic the natural conditions in calcareous soils. Leaf chlorophyll concentration was estimated every 3–4 days using the portable instrument SPAD-502 meter. Chlorophyll fluorescence parameters, photosynthetic capacity estimated from oxygen evolution, leaf Fe concentrations, and root tip ferric chelate reductase activity were measured at the end of the experiment. Plants from the 0 and 5 μM Fe treatments showed leaf chlorosis and had decreased leaf chlorophyll concentrations. Leaves of plants grown in the absence of Fe in the solution had smaller rates of oxygen evolution both in the presence and absence of calcium carbonate, compared with plants grown in the presence of 10 μM Fe. In the absence of calcium carbonate the photosystem II efficiency, estimated from fluorescence parameters, was similar in all treatments. A slight decrease in photosystem II efficiency was observed in plants grown without Fe and in the presence of calcium carbonate. A 2.5-fold increase in root tip ferric chelate reductase activity over the control values was found only when plants were grown with low levels of Fe and in the presence of calcium carbonate.     

Repression of the 14-3-3 gene affects the amino acid and mineral composition of potato tubers

Recently, transgenic potato plants were created showing underexpression of the 20R isoform of the 14-3-3 protein. The transgenic plants grown in tissue culture showed a significant increase in nitrate reductase activity and a decrease in nitrate level. The transgenic line with the lowest 14-3-3 quantity was field-trialed (1997-2000) and analyzed. The reduction in the 14-3-3 protein level consistently resulted in a starch content increase and in an increase in the ratio of soluble sugars to starch in the tubers, although the latter was only barely visible. The determination of amino acid composition in the tubers showed a significant increase in methionine, proline, and arginine content and a slight but consistent increase in hydrophobic amino acid and lysine content in the cells of the transgenic potato plants. We also observed an increase in the crude protein content, from 19 to 22.1% of the control value in consecutive years. It is proposed that all of these changes might have resulted from the downregulation of nitrate reductase and sucrose phosphate synthase activities by 14-3-3, although other potential mechanisms cannot be excluded (e.g., an increase in enzyme protein level). 14-3-3-repressed transgenic plants showed a significant increase in calcium content in their tubers. It is thus proposed that a function of the isolated 14-3-3 isoform is in the control of amino acid synthesis and calcium metabolism. However, the mechanism of this control is as yet unknown.     

Involvement of programmed cell death in suppression of the number of root nodules formed in soybean induced by Bradyrhizobium infection

Legumes establish symbiosis with nitrogen-fixing rhizobia through root nodules to acquire nitrogen. Legumes control nodule number through systemic (autoregulation of nodulation) as well as local regulation. Moreover, plants defend themselves against bacteria and other pathogens through the induction of localized (localized acquired resistance) and systemic (SAR, systemic acquired resistance; ISR, induced systemic resistance) responses. Herein, we show that the number of root nodules is suppressed by programmed cell death (PCD), and is simultaneously controlled by SAR and ISR in soybean (Glycine max [L.] Merr.). The wild-type soybean cultivar Williams 82 showed markedly fewer root nodule primordia and PCD symptoms, including accelerated DNA degradation, enhanced generation of reactive oxygen species (visualized by 3,3′-diaminobenzidine staining), and excessive cell death (detected on staining with trypan blue) compared to the hypernodulation mutant NOD1-3. These results suggest that PCD suppresses the formation of root nodules in wild-type soybean. In addition, microarray and gene ontology analyses showed that essential components of hypersensitive response (HR) or disease resistance, such as resistance (R) genes, mitogen-activated protein kinase cascade, SAR, salicylic acid, jasmonic acid, ethylene, etc., were activated in wild-type plants. These analyses corroborate the above findings, demonstrating that the suppression of root nodule formation by PCD is accompanied by HR, and is simultaneously controlled by SAR and ISR in soybean. These findings provide new insight into the control of nodulation to balance nutritional requirements and energy status in legumes.     

EFFECT OF NITRATE NUTRITION ON GROWTH AND NITROGEN ASSIMILATION OF PEARL MILLET EXPOSED TO SODIUM CHLORIDE STRESS

Influence of nitrate concentration on growth and nitrogen assimilation in salinity-stressed pearl millet (Pennisetum typhoids L.) was studied. The plants were grown in perlite and irrigated with nutrient solution containing 0, 25, 50, or 100 mM sodium chloride (NaCl) in the presence of 2 or 10 mM NO₃ ^?. Salinity decreased leaf dry weight and soluble proteins, as well as total chlorophyll. Free amino acid content, including proline, was higher in salt-stressed plants compared to controls. The activities of nitrate reductase, nitrite reductase, and glutamate synthase were reduced, but the glutamine synthetase activity was less affected. High nitrate (10 mM) in the irrigation solution partially restored activities of the above enzymes and increased the soluble protein content despite the high NaCl concentration. The retarded growth of pearl millet due to salinity stress was partially restored in the presence of high nitrate concentration in the irrigation solution.     

Urea as an organic nitrogen source for hydroponically grown tomatoes in comparison with inorganic nitrogen sources

Tomato (Lycopersicon esculentum Mill., cv. Momotaro) plants were grown in nutrient solutions with several levels of urea, nitrate, and ammonium alone or in combination to evaluate the role of urea as an organic nitrogen source compared with that of nitrate and/or ammonium as inorganic nitrogen sources. Nitrogen deficiency and excess symptoms were detected in the urea-fed plants at lower (28 mg N L^-1) and higher nitrogen levels (336, 504 mg N L^-1), respectively. The effect of urea on plant growth and leaf elemental composition was intermediate between that of nitrate and ammonium. Solution pH under urea nutrition slightly increased or remained stable. When plants were cultured with the solution containing 168 mg N L^-1, the total dry weight of the plants which received urea+nitrate was significantly higher than that of the plant which received urea and was almost equal to that of the plants which received nitrate or nitrate+ammonium. Both absorption and utilization of nitrogen in the plants fed with urea decreased compared with those of the plants fed with nitrate or ammonium. The insufficient absorption and utilization of nitrogen were estimated to be the main factors associated with the growth reduction of tomato plants under urea nutrition. However, combined application of urea and nitrate is useful for adequate plant growth without a reduction of the cation absorption in tomato while maintaining a stable solution pH.     

硝态氮抑制尖孢镰刀菌侵染促进黄瓜生长的内在生理机制

  【目的】  连作障碍严重影响设施农业的发展。不同形态氮素可影响黄瓜土传枯萎病的发生,然而其内在生理机制尚不明确。通过氮素营养调控植物–微生物互作关系,为防控土传病害的发生提供理论依据。  【方法】  以黄瓜品种津春2号和尖孢镰刀菌黄瓜专化型菌 (FOC) 为试材,进行温室营养液培养试验。设营养液中添加铵态氮不接菌 (A)、硝态氮不接菌 (N)、铵态氮接菌 (AI) 和硝态氮接菌 (NI) 共4个处理。尖孢镰刀菌侵染8天后进行植株样品的采集及测定,包括株高、根长、生物量、病情指数、叶绿素含量、光合特性、叶片温度,并进行了叶肉细胞超微结构的观察,测定了植物全氮、可溶性蛋白及可溶性糖含量。  【结果】  与铵态氮相比,硝态氮营养显著抑制了黄瓜植株枯萎病的发病率,并显著促进了植株的生长以及植株生物量的增加。未接菌条件下,供应铵态氮的植株光合速率、气孔导度、蒸腾速率、羧化效率及表观量子效率均显著高于供应硝态氮的植株;尖孢镰刀菌的侵染导致供应铵态氮的植株叶绿体结构受损,显著降低了其光合速率、气孔导度、蒸腾速率、细胞间隙CO₂浓度、羧化效率及表观量子效率,而病原菌侵染对供应硝态氮的植株叶片光合特性无显著影响。未接菌条件下,供应铵态氮的植株叶片温度及水分利用效率显著低于供应硝态氮的植株;尖孢镰刀菌侵染后,供应铵态氮的植株叶片温度及水分利用率显著增加,而病原菌侵染对供应硝态氮的植株无显著影响。叶片温度与蒸腾速率呈显著负相关关系,而与水分利用率呈显著正相关关系。供应铵态氮的植株根系全氮、可溶性蛋白及可溶性糖含量均显著高于供应硝态氮的植株,从而促进病原菌对供应铵态氮的植株的侵染。尖孢镰刀菌侵染后,供应铵态氮的植株根系可溶性蛋白含量显著增加,可溶性糖含量降低,而尖孢镰刀菌侵染对供应硝态氮的植株可溶性蛋白及可溶性糖含量无显著影响。  【结论】  硝态氮能够有效地抑制黄瓜枯萎病的发生,维持叶绿体结构的完整性,保持黄瓜植株正常的光合作用及生长,并减少碳水化合物向根系的运输,从而抑制病原菌的侵染及病害的发生。在黄瓜的设施栽培中,可适当增加硝态氮肥的施用而减少铵态氮肥的投入,以抑制土传枯萎病发生。     

Effects of root-zone acidity on utilization of nitrate and ammonium in tobacco plants

Tobacco (Nicotiana tabacum L., cv. 'Coker 319') plants were grown for 28 days in flowing nutrient culture containing either 1.0 mM NO3- or 1.0 mM NH4+ as the nitrogen source in a complete nutrient solution. Acidities of the solutions were controlled at pH 6.0 or 4.0 for each nitrogen source. Plants were sampled at intervals of 6 to 8 days for determination of dry matter and nitrogen accumulation. Specific rates of NO3- or NH4+ uptake (rate of uptake per unit root mass) were calculated from these data. Net photosynthetic rates per unit leaf area were measured on attached leaves by infrared gas analysis. When NO3- [correction of NO-] was the sole nitrogen source, root growth and nitrogen uptake rate were unaffected by pH of the solution, and photosynthetic activity of leaves and accumulation of dry matter and nitrogen in the whole plant were similar. When NH4+ was the nitrogen source, photosynthetic rate of leaves and accumulation of dry matter and nitrogen in the whole plant were not statistically different from NO3(-) -fed plants when acidity of the solution was controlled at pH 6.0. When acidity for NH4(+) -fed plants was increased to pH 4.0, however, specific rate of NH4+ uptake decreased by about 50% within the first 6 days of treatment. The effect of acidity on root function was associated with a decreased rate of accumulation of nitrogen in shoots that was accompanied by a rapid cessation of leaf development between days 6 and 13. The decline in leaf growth rate of NH4(+) -fed plants at pH 4.0 was followed by reductions in photosynthetic rate per unit leaf area. These responses of NH4(+) -fed plants to increased root-zone acidity are characteristic of the sequence of responses that occur during onset of nitrogen stress.     

Interactions of bacteria,protozoa and plants leading to mineralization of soil nitrogen

The capacity of bacteria and protozoa to mineralize soil nitrogen was studied in microcosms with sterilized soil with or without wheat plants. The effect of small additions of glucose or ammonium nitrate or both, twice a week was also tested. Plant dry weight and N-content, number of microorganisms and biomass plus inorganic N were determined after 6 weeks.The introduction of plants profoundly influenced the N transformations. In the presence of root-derived carbon, much more N was mineralized from the organic matter and immobilized mainly in plant biomass. “Total observable change in biomass N plus inorganic N” was negative in the unvegetated soils without additions, while a mineralization of 1.7 mg N microcosm^?1 was observed in microcosms with wheat plants grown with bacteria only. When protozoa were included, the N taken up by plants increased by 75%. Sugar additions resulted in an 18% increase of total N in the shoots when protozoa were present, but had no significant effect in the absence of grazers. Plants with the same root weight were more efficient in their uptake of inorganic N when protozoa were present. Plants grown with protozoa also had a lower R/S ratio, indicating a less stressed N availability situation. The lowest ratio was found with N additions in the presence of protozoa.The results indicate that, with energy supplied by plant roots or with external glucose additions, soil bacteria can mineralize N from the soil organic matter to support their own growth. Grazing of the bacteria is necessary to make bacterial biomass N available for plant uptake.     

Growth attributes,xylem sap composition,and photosynthesis in common bean as affected by nitrogen and phosphorus deficiency

The effects of nitrogen (N‐) and phosphorus (P‐) deficiency, isolatedly or in combination, on growth, nitrogenous fraction, and inorganic phosphate in xylem exudade, and photosynthesis of common bean (Phaseolus vulgaris L. cv. Negrito) were investigated. Plants were grown in nutrient solution adjusted daily to pH 5.5 and aerated continuously. Ten days after emergence mineral deficiency was imposed. Plants were then supplied with high N (7.5 mol m^‐3) or low N (0.5 mol m^‐3), and also with high P (0.5 mol m^‐3) or low P (0.005 mol m^‐3). All sampling and measurements were made 28 days after emergence. N‐ or P‐deprivation brought about large decreases in total leaf area by inhibiting the emergence of new leaves and primarily the expansion of the leaves. The specific leaf area did not change under N‐ but decreased under P‐limitation. The decreased shoot to root ratio in all deficiency treatments was a consequence of a lowering mass of above‐ground organs, especially of leaves.

The content of chlorophylls declined significantly only under N‐deficiency alone; carotenoids declined under both N‐ and combined N‐ and P‐limitation. No alteration in amino acid concentration in xylem exudate occurred in plants experiencing N‐starvation, while ureides increased by 79%, and nitrate and inorganic phosphate decreased greatly. Under P‐deprivation, amino acids and nitrate in xylem sap dropped by about half; ureides were held relatively constant, and phosphate was severely depressed. Total upward translocation of N through xylem was estimated to be about 16% higher in N‐deficient plants than in plants without mineral limitation, but leaf N levels in the former were lower as compared to control plants. The net carbon (C) assimilation decreased similarly regardless of the imposed deficiency treatment. Such a decrease was mainly determined by non‐stomatal factors. In general, no additive effect between N‐ and P‐limitation on any of measured parameters was observed.     

Effect of simulated acid rain on the growth of soybean

Soybean seedlings (Glycine max) grown in a glasshouse were exposed to simulated acid rain using a solution of deionized water containing sulfate, nitrate and chloride in concentrations and proportions equivalent to those in ambient rain water. Plants were subjected to acid rain treatment twice a week during the growing season, for a 1 hr period at a rate of 5 mm hr^?1. When the acid rain was below pH 3.0, visible symptoms developed in the young trifoliate leaves. However, at a pH above 3.0 there was no evidence of visible leaf injury; also tissue dry weights and leaf areas were not affected even after 7 weeks of exposure. The number of root nodules in plants exposed to acid rain at pH 4.0 tended to be higher than those of control plants maintained at pH 5.6, but decreased subsequently with decreasing pH. Based on our results current ambient levels of rain acidity in Japan should not have an adverse impact on seedling growth in soybean.     

Effect of potassium on growth and the electrophoretic pattern of leaf proteins of sunflower supplied with ammonium or nitrate

Sunflower (Helianthus annuus L.) was grown on liquid medium containing either nitrate, ammonium or ammonium + nitrate, with or without potassium. The growth of plants supplied with nitrate or ammonium + nitrate in the presence of potassium (5 mmol/l) was comparable. Plants grown on ammonium‐N only showed significantly lower growth. Dry matter and organic nitrogen content in plants supplied with ammonium + nitrate (total nitrogen 10 mmol/1) in the presence of potassium (5 mmol/l) was higher than in nitrate grown plants. Leaf protein pattern on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) was not affected by the nitrogen source provided in the medium.

Potassium deficiency led to reduced growth, necrotic spots on the leaves and specific alterations in leaf protein pattern, expressed by an increase or decrease in several polypeptides. This was common to all the nitrogen forms tested. A most pronounced change was the increased expression of polypeptides of molecular weight 60 and 62 kilodaltons.