Effect of head removal on leaf senescence of sunflower.

Greenhouse and field studies examined the effect of flower or seedhead removal on leaf senescence and associated changes in sunflower (Helianthus annuus L.) plants. At intervals during seed development, selected leaves (leaves 6 through 8 from the top in the greenhouse and leaf 7 from the top in the field) were harvested and analyzed for chlorophyll, specific leaf weight, N, P, soluble protein, and electrophoretic gel profiles of soluble polypeptides. In both the greenhouse and the field, the leaves of headless plants retained or accumulated more N, P, soluble protein, and dry weight than leaves of plants with heads. Obviously, head removal affected the partitioning of these metabolites during seed development. None of the treatments resulted in the formation of new polypeptides (electrophoretic gel profiles). Comparisons of the rates and extent of loss of chlorophyll, soluble protein, and polypeptide bands (especially ribulose 1,5-bisphosphate carboxylase) from the leaves of headed and deheaded plants showed that head removal delayed the rate of development of leaf senescence for the greenhouse-grown but had much less effect on field-grown plants. These findings illustrate the variability in different parameters commonly associated with the leaf senescence processes of headed and deheaded sunflower plants grown under different environments.

Effect of foliar applications of urea on accelerated senescence of maize induced by ear removal.

Field grown maize (Zea mays L. cv B73 x Mo17) plants, with and without ears, were sprayed with urea solutions to determine whether foliar application of N could prevent or delay the accelerated loss of reduced N from the leaf and leaf senescence induced by ear removal. Urea sprays were applied at 7, 14, and 21 days after anthesis in three separate and equal applications that provided a total of 67 kilograms N per hectare or 1 gram N per plant. Treatments were arranged in a 2 x 2 factorial in a randomized complete block with five replicates. Appropriate plant and leaf samplings and assays were made.In response to spray treatments, net increases of reduced N were detected in the whole shoot and plant parts, especially the stalk of the earless plants and grain of the eared plants. There was no effect of urea spray treatment on the normal loss of N from the leaves or rate of senescence of the eared plants or on the accelerated loss of N from the leaves or rate of senescence induced by ear removal. Grain and stover yields were unaffected by the spray treatment.Apparently the plants were unable to utilize the urea N applied to the vegetation (primarily leaves) after anthesis to enhance or extend the accumulation of dry weight by either eared or earless plants.

Effect of nodulation on assimilate remobilization in soybean.

The objectives of this work were to determine the effect of nodulation on dry matter, reduced-N, and phosphorus accumulation and partitioning in above-ground vegetative parts and pods of field-grown soybean (Glycine max [L.] Merr. cv Harosoy).From comparison of nodulated and nonnodulated isolines, it was estimated that nodulated plants attained 81 and 71% of total-plant (above ground) N from uptake of soil N in 1981 and 1982, respectively. These data, along with visibly greener leaves of nodulated plants, led us to assume that nonnodulated plants were under a moderate N stress relative to nodulated plants. Nonnodulated plants accumulated less total-plant N and partitioned less dry matter and N to the pods, compared with nodulated plants. This occurred even though net photosynthesis, as estimated by rate and amount of dry matter accumulation, was the same for both nonnodulated and nodulated plants. Rate of dry matter and reduced-N accumulation in pods was less for nonnodulated than for nodulated plants while duration of podfill was similar for both isolines. From these data we concluded that moderate N stress affected partitioning of photosynthate rather than net photosynthesis, and that N played a role in translocation of photosynthate to the pods. Total plants (above-ground portion) and pods of both nodulated and nonnodulated plants accumulated similar amounts of phosphorus, which indicated that phosphorus and N accumulation were independent.Remobilization of nitrogen and phosphorus from vegetation to pods preceded dry matter remobilization. It appeared that either more nitrogen accumulation prior to podfill, or continued nitrogen assimilation during podfill would increase nitrogen and dry matter partitioning to pods, but that increasing photosynthesis without concomitantly increasing nitrogen input may not necessarily result in enhanced seed production.

Acetaldehyde Oxime, A Product Formed during the In Vivo Nitrate Reductase Assay of Soybean Leaves.

Evolution of nitrogen oxides (NO((x)), primarily as nitric oxide) from soybean (Glycine max [L.] Merr.) leaves during purged in vivo nitrate reductase assays had been reported; however, these reports were based on a method that had been used for determination of NO((x)) in air. This method also detects other N compounds. Preliminary work led us to doubt that the evolved N was nitric oxide. Studies were undertaken to identify the N compound evolved from the in vivo assay that had been reported as NO((x)). Material for identification was obtained by cryogenic trapping and fractional distillation, and by chemical trapping procedures. Mass spectrometry, ultraviolet spectroscopy, and (15)N-labeled nitrate were used to identify the compounds evolved and to determine whether these compounds were derived from nitrate. Acetaldehyde oxime was identified as the predominant N compound evolved and this compound is readily detected by the method for NO((x)) determination. Substantial quantities of acetaldehyde oxime (16.2 micromoles per gram fresh weight per hour) were evolved during the in vivo assay. Small amounts of nitrous oxide (0.63 micrograms N per gram fresh weight per hour) were evolved, but this compound is not detected as NO((x)). Acetaldehyde oxime and nitrous oxide were both produced as a result of nitrate ((15)NO(3) (-)) reduction during the assay.

Effects of Pod Removal on Metabolism and Senescence of Nodulating and Nonnodulating Soybean Isolines: II. Enzymes and Chlorophyll.

The objectives of this work were to determine the effect of sink strength (presence or absence of pods) and nitrogen source (nodulating versus nonnodulating plants) on enzymic activities, chlorophyll concentration, and senescence of soybean (Glycine max [L.] Merr. cv Harosoy) isolines. A 2-year (1981-1982) field study was conducted.For both nodulated and nonnodulated plants, ribulose bisphosphate carboxylase (RuBPCase) activity of upper-canopy leaves was decreased by pod removal in both years, while chlorophyll concentration was decreased in 1981 only. Nonnodulated plants had lower RuBPCase activity in 1981 and lower chlorophyll concentration in both years compared with nodulated plants. In both years, and for all treatments, RuBPCase activity and chlorophyll began to decline at about the same time, but the rate of decline was less for depodded than for podded plants. Leaves in the middle and lower parts of the canopy had similar RuBPCase activity and chlorophyll concentration trends as upper-canopy leaves for all treatments.Profiles of nitrate reductase activity (NRA) were similar for all treatments in both 1981 and 1982. Acetylene reduction profiles were similar for nodulated-podded and nodulated-depodded plants. The peak and decline in NRA profiles preceded the peak and decline in acetylene reduction profiles. The rate of decline in acetylene reduction activity was less for depodded plants, especially in 1982, but activities reached zero by the final sampling time. Thus, nodule senescence was not prevented by pod removal.Based on seasonal profiles of RuBPCase activity, chlorophyll, NRA, and acetylene reduction activity, the initiation of senescence appeared to occur at the same approximate time for all treatments and, thus, did not depend on the presence or absence of pods or nodules. The hypothesis that nodules act as a nitrogen source and carbohydrate sink to delay senescence in the absence of pods was not correct.

Effects of pod removal on metabolism and senescence of nodulating and nonnodulating soybean isolines: I. Metabolic constituents.

Field studies were conducted in 1981 and 1982 to ascertain the effects of pod removal on senescence of nodulating and nonnodulating isolines of soybean (Glycine max [L.] Merr. cv Harosoy) plants. Specifically, the test hypothesis was that nodules act as a nitrogen source and a carbohydrate sink which would in turn prevent or delay senescence in the absence of pods. Senescence was judged by changes in metabolite levels, in dry matter accumulation, and by visual observation.For both nodulated and nonnodulated plants, pod removal had no effect on the magnitude or rate of dry matter and reduced-N accumulation by whole plants. Phosphorus accumulation was significantly less in both nodulated- and nonnodulated-depodded plants, compared with respective control plants with pods. These data suggested a role for pods in phosphorus uptake. Accumulation of dry matter, reduced N, and phosphorus ceased at approximately the same time for all treatments.Pod removal did affect partitioning of plant constitments, with leaves and stems of depodded plants serving as a major alternate sink for accumulation of dry matter, reduced N, phosphorus, and nonstructural carbohydrates (primarily starch). While depodded plants eventually lost a significant amount of leaves, leaf drop was delayed relative to plants with pods; and depodded plants still retained some green leaves at 2 weeks past grain maturity of control (podded) plants.The results indicated that senescence patterns of soybean plants were the same for nodulated and nonnodulated plants, and that pods did not control the initiation of senescence, but rather altered the partitioning of plant constituents and the visual manifestations of senescence.

Differential Senescence of Maize Hybrids following Ear Removal : I. Whole Plant.

Visual senescence symptoms and associated changes in constituent contents of three field-grown maize (Zea mays L.) hybrids (Pioneer brand 3382, B73 x Mo17, and Farm Service brand 854) were compared in response to ear removal. Whole plants were harvested at eight intervals during the grain-filling period, and analyzed for dry matter, total N and nitrate N, phosphorus, sugars, and starch.Upper leaves of earless P3382 and B73 x Mo17 showed reddish discoloration by 25 days after anthesis (DAA) and all leaves had lost most of their chlorophyll by 40 DAA. In striking contrast, leaves of earless FS854 plants remained green and similar in appearance to eared controls throughout the grain-filling period.For all hybrids, ear removal led to a decrease in dry weight, reduced N, total N, and phosphorus contents of the total plant, and an increase in carbohydrate content of the leaves and stalks, relative to respective controls. Although changes in carbohydrate and N contents, which previously had been associated with senescence, were observed for all earless hybrids, these changes were followed by accelerated senescence and early death only for P3382 and B73 x Mo17. By 30 DAA, earless P3382 and B73 x Mo17 plants ceased to accumulate dry weight, total N, and phosphorus, indicating a termination of major metabolic activities. In contrast, earless FS854 plants retained a portion of these metabolic activities until 58 DAA, indicating a role for roots in determining rate of senescence development. Thus, the course of senescence was more accurately reflected by measurements of metabolic activities than by measurements of metabolite contents at any given time. These results show that the ear per se does not dictate the rate or completion of the senescence process, and implicated an association between the continued accumulation of N and associated root activities with the delayed senescence pattern of the earless FS854 plants. It is evident that studies involving control of senescence among species must also consider genotypic influences within species.

Differential Senescence of Maize Hybrids following Ear Removal : II. Selected Leaf.

In conjunction with a study of the effects of ear removal on the senescence of whole maize (Zea mays L.) plants, visual symptoms and associated changes in constituent contents and activities of a selected leaf (first leaf above the ear) were determined. Leaves were sampled from field-grown eared and earless Pioneer brand 3382, B73 x Mo17, and Farm Services brand 854 maize hybrids at nine times during the grainfilling period.VISUAL SYMPTOMS INDICATED THE FOLLOWING SEQUENCE AND RATE OF SENESCENCE: earless B73 x Mo17 > earless P3382 >> eared B73 x Mo17 >> eared P3382 eared FS854. All earless hybrids showed increases in leaf dry weight and sugar content; however, the increases were transitory for P3382 and B73 x Mo17, but continuous throughout the grain-filling period for FS854, indicative of continued photosynthetic activity of the latter. All earless hybrids exhibited similar and transitory starch accumulation patterns. Thus, FS854 was an exception to the concept that carbohydrate accumulation accelerates leaf senescence. Ear removal resulted in accelerated losses of reduced N, phosphoenolpyruvate and ribulose bisphosphate carboxylases, phosphorus, chlorophyll, nitrate reductase activity, and moisture for P3382 and B73 x Mo17 plants. In contrast, the loss of all components (except phosphorus) was similar for the selected leaf of earless and eared FS854.Although the loss of nitrate reductase activity, reduced N, and carboxylating enzymes accurately reflected the development of senescence of the selected leaf, the rate of net loss of reduced N and carboxylating enzymes appeared to be regulated. We deduced that the rate of flux of N into the leaf was a factor in regulating the differing rates of senescence observed for the six treatments; however, we cannot rule out the possibility of concurrent influence of growth regulators or other metabolites.

Metabolism of carbon and nitrogen by soybean seedlings in response to vegetative apex removal.

Short-term (31-hour diurnal) growth-chamber studies were conducted to determine the effects of removing the vegetative apex (meristem and developing trifoliolate leaves) on net photosynthesis (changes in plant dry weight), on distribution of metabolites among plant parts, and on nitrate metabolism and reduced-N accumulation by soybean [Glycine max (L.) Merr.] seedlings. Roots and stems served as alternate sinks for dry matter accumulation in the absence of the vegetative apex. Sugar concentration in roots increased (42%) within 4 hours of vegetative apex removal, and remained higher than for the controls during the 31-hour experimental period. Nitrate assimilation (nitrate reductase activity and total accumulation of reduced-N) was also enhanced in response to vegetative apex removal. Although dry matter accumulation was similar between treated and control plants (113 versus 116 milligrams per plant) over the 31-hour sampling period, more nitrate (1.31 versus 0.79 milligrams per plant) and more reduced-N (3.96 versus 3.45 milligrams per plant) accumulated in treated plants during the same interval. It was concluded that vegetative apex removal had little effect on overall net photosynthesis of soybean seedlings during the 31-hour treatment period, but did alter partitioning of photosynthate and enhanced uptake, transport, and reduction of nitrate. Implications are that uptake and metabolism of nitrate by soybeans may be limited by flux of carbohydrate to the roots, although hormonal effects due to vegetative apex removal cannot be ruled out.

Assimilation of [N]Nitrate and [N]Nitrite in Leaves of Five Plant Species under Light and Dark Conditions.

Light dependency of nitrate and nitrite assimilation to reduced-N in leaves remains a controversial issue in the literature. With the objective of resolving this controversy, the light requirement for nitrate and nitrite assimilation was investigated in several plant species. Dark and light assimilation of [(15)N]nitrate and [(15)N]nitrite to ammonium and amino-N was determined with leaves of wheat, corn, soybean, sunflower, and tobacco. In dark aerobic conditions, assimilation of [(15)N]nitrate as a percentage of the light rate was 16 to 34% for wheat, 9 to 16% for tobacco, 26% for corn, 35 to 76% for soybean, and 55 to 63% for sunflower. In dark aerobic conditions, assimilation of [(15)N]nitrite as a percentage of the light rate was 11% for wheat, 7% for tobacco, 13% for corn, 28 to 36% for soybeans, and 12% for sunflower. It is concluded that variation among plant species in the light requirement for nitrate and nitrite assimilation explains some of the contradictory results in the literature, but additional explanations must be sought to fully resolve the controversy.In dark anaerobic conditions, the assimilation of [(15)N]nitrate to ammonium and amino-N in leaves of wheat, corn, and soybean was 43 to 58% of the dark aerobic rate while dark anaerobic assimilation of [(15)N]nitrite for the same species was 31 to 41% of the dark aerobic rate. In contrast, accumulation of nitrite in leaves of the same species in the dark was 2.5-to 20-fold higher under anaerobic than aerobic conditions. Therefore, dark assimilation of nitrite cannot alone account for the absence of nitrite accumulation in the in vivo nitrate reductase assay under aerobic conditions. Oxygen apparently inhibits nitrate reduction in the dark even in leaves of plant species that exhibit a relatively high dark rate of [(15)N]nitrite assimilation.

Efficiency of Nitrogen Assimilation by N(2)-Fixing and Nitrate-Grown Soybean Plants (Glycine max [L.] Merr.).

Nodulated and non-nodulated (not inoculated) soybeans (Glycine max [L.] Merr. cv Wells) were grown in controlled environments with N(2) or nonlimiting levels of NO(3) (-), respectively, serving as sole source of nitrogen. The efficiency of the N(2)-fixing plants was compared with that of the nitrate-supplied plants on the basis of both plant age and plant size. Efficiency evaluations of the plants were expressed as the ratio of moles of carbon respired by the whole plant to the moles of nitrogen incorporated into plant material.Continuous 24-hour CO(2) exchange measurements on shoot and root systems made at the beginning of flowering (28 days after planting) indicated that N(2)-fixing plants respired 8.28 moles of carbon per mole of N, fixed from dinitrogen, while nitrate-supplied plants respired only 4.99 moles of carbon per mole of nitrate reduced. Twenty-one-day-old nitrate-supplied plants were even more efficient, respiring only 3.18 moles of carbon per mole of nitrate reduced. The decreased efficiency of the N(2)-fixing plants was not due to plant size since, on a dry weight basis, the 28-day-old N(2)-fixing plants were intermediate between the 28- and 21-day-old nitrate-supplied plants.The calculated efficiencies were predominantly a reflection of root-system respiration. N(2)-fixing plants lost 25% of their daily net photosynthetic input of carbon through root-system respiration, compared with 16% for 28-day-old nitrate-supplied plants and 12% for 21-day-old nitrate-supplied plants. Shoot dark respiration was similar for all three plant groups, varying between 7.9% and 9.0% of the apparent photosynthate.The increased respiratory loss by the roots of the N(2)-fixing plants was not compensated for by increased net photosynthetic effectiveness. Canopy photosynthesis expressed on a leaf area basis was similar for 28-day-old N(2)-fixing plants (15.5 milligrams CO(2) square decimeter per hour) and 21-day-old nitrate-supplied plants (14.5 milligrams CO(2) square decimeter per hour). Both were similar in total canopy leaf area. The larger nitrate-supplied plants (28-day-old) had lower photosynthetic rates (12.5 milligrams CO(2) square decimeter per hour), presumably due to self-shading of the leaves.These data indicate that, during the early stages of plant development, dependence solely on N(2)-fixation is an expensive process compared to nitrate reduction in nitrate-supplied plants, since the N(2)-fixing plants retained 8% to 12% less of their photosynthate as dry matter.

Interaction of carbon and nitrogen metabolism in the productivity of maize.

Five maize (Zea mays L.) hybrids, FS854, B73 x Mo17, B84 x Mo17, B73 x B77, and P3382, grown under field conditions, were sampled at intervals during the grain-filling period. Plants were subdivided into stalks (including sheaths), leaves, and kernels. These parts were assayed for dry weight, reduced nitrogen, and extractable nonstructural carbohydrates. The duration and rates of net nitrate reduction and photosynthesis were approximated by the changes over time in the accumulation of reduced nitrogen and dry weight by the plant (total, above ground), respectively.Data on the accumulation of reduced nitrogen and dry weight by the plant show that decreases in nitrate reduction preceded (in time and extent for four of the hybrids and in extent for FS854) decreases or cessation of photosynthesis. FS854 continued to accumulate reduced nitrogen and dry matter throughout the grain-filling period.The patterns of change in stalk carbohydrate and reduced nitrogen during the early stages of ear development show the stalk serves as a storage reservoir and that these reserves were remobilized during the final stages of grain development. The marked increase and maintenance of dry weight and carbohydrate content of stalks until 34 days after anthesis, shows the capacity of the leaves to produce photosynthate through the first half of the grain-filling period exceeds the needs of the ear and/or the transport system. In contrast, stalk nitrogen content shows a slight increase up to 12 days after anthesis and decreases continually thereafter. Leaf nitrogen was lost continuously throughout grain development. The potential capacity of the plant to supply newly reduced nitrogen was inadequate to support initiation and early development of the kernels without remobilization of vegetative nitrogen. Of the two hybrids having delayed leaf senescence, FS854 with its initially higher concentration and content of reduced nitrogen in the stalk, initiated and developed a bigger ear than P3382, which had lower levels of stalk nitrogen.Three of the five hybrids had ;near linear' rates of accumulation of kernel dry weight, whereas none of the hybrids had linear rates of gain in kernel nitrogen. All hybrids had maximum or near maximum rates of gain of kernel nitrogen between 26 and 34 days after anthesis and a marked reduction (41-52%) of rates in the following sampling interval. These decreases are concurrent with decreases in rates of nitrate reduction (nitrogen accumulation) by the whole plant for four of the hybrids and with decreases in remobilization of nitrogen from the vegetation of FS854. Data for the ratio of rates of accumulation of dry weight/reduced nitrogen by the kernels versus time after anthesis, show that the accumulation of dry weight and reduced nitrogen are independent of each other. The variations in the ratio values appear best related to variations in the availability of nitrogen from the vegetation.

The effects of ear removal on senescence and metabolism of maize.

Ears were removed from field grown maize (Zea mays L.) to determine the effects on senescence and metabolism and to clarify conflicting literature reports pertaining to these effects. Ears were removed at three days after anthesis and comparisons were made of changes in metabolism between eared and earless plants until grain of the eared plants matured as judged by black layer formation.The initial visual symptom following ear removal was the development of reddish colored leaves. As judged by leaf yellowing, the removal of ears not only initiated an earlier onset but enhanced the rate of senescence. With this exception, the visual patterns of senescence were similar for earless and eared plants. Other characteristics associated with ear removal were: (a) marked decrease in dry weight and reduced N accumulation by the whole plant; (b) progressive, parallel decreases in leaf reduced N, nitrate reductase activity, and chlorophyll; (c) increases in carbohydrate content of both the leaf and stalk and of reduced N in the stalk. These changes indicate that ear removal reduced photosynthesis and nitrate reduction by approximately equal proportions and that the stalk serves as an alternate sink for both carbohydrate and nitrogen.The remobilization of nitrogen from the leaf was not dependent on the presence of an ear. A logical reason for the more rapid loss of nitrogen from the leaf of the earless plants appears to be the cessation of nitrate uptake and/or flux of nitrate to the leaves.From these results and from related experiments we tentatively conclude that the loss of nitrogen from the leaf is a major cause of death of the intact maize plant.

Availability of reduced N and carbohydrates for ear development of maize.

Changes in dry weights, reduced N, nitrate, and nitrate reductase activity of various plant parts of the above ground vegetation (stover) and ears of field grown maize were measured at intervals between anthesis and grain maturity. Nonstructural carbohydrate contents were also measured in some instances. Changes in dry weight and reduced N content were used to approximate net in situ photosynthetic and nitrate assimilation activities and to determine whether the availability of photosynthate or reduced N was limiting grain production.Of the five hybrids studied, all showed extensive remobilization (loss) of reduced N from the stover during grain development. This loss of stover N was initiated by 18 to 21 days after anthesis. Most of this loss of N (about 70%) was from the leaves. In contrast, three of the five hybrids had more vegetative dry weight at grain maturity than at anthesis, while the loss of stover dry weight by the other two hybrids was negligible. By 42 days after anthesis when the bulk of the ear weight had been acquired, the average gain in stover dry weight for the five hybrids was 12% while the loss of stover reduced N was 28%. Where measured, the increase in stover dry weight was largely due to deposition of carbohydrates in the stalk. These results show that the photosynthetic capacity was adequate while nitrate reduction capacity was inadequate for ear demands. The changes in the rate of accumulation of dry weight and reduced N by the ear indicated that the rate of supply of reduced N to the ear could have limited ear development for one of the five hybrids. The dry weight and carbohydrate (where measured) accumulation in the vegetation during the first 42 days after anthesis infers that the rate of supply of photosynthate to the ear was probably not a limiting factor for any of the five hybrids.The maximum remobilization of stover N during grain development was 1.8 g N plant(-1) for the genotypes examined, while the amount of reduced N accumulated by the grain varied from 1 to 5 g plant(-1). The amount of newly reduced N (nitrate reduced after anthesis) provided from 48 to 72% of the total N accumulated by the ear. The relative amounts of newly reduced N and remobilized N vary with genotype and environment. With respect to insuring high productivity, it was concluded that there is more flexibility in the system (genotype and environment) for increasing the supply of newly reduced N than in enhancing the remobilization of vegetative N.

Relationship between Nitrate Uptake, Flux, and Reduction and the Accumulation of Reduced Nitrogen in Maize (Zea mays L.): I. GENOTYPIC VARIATION.

The study presented here was an extension of a preceding field project concerned with changes in N metabolism of four maize hybrids during grain development. The objectives were to relate uptake, flux, and reduction of nitrate to accumulation of reduced N in growth-chamber-grown seedlings of the same four hybrids and to compare these results with those obtained in the field study.Hybrid D took up more nitrate than the other three hybrids, primarily because of a larger root system. The correlations between total N (nitrate plus reduced N plant(-1)) accumulated by harvest and root dry weight or shoot to root ratios were r = +0.97 and -0.90, respectively. Correlations with shoot dry weight were low. Although the larger root system indicates enhanced partitioning of photosynthate to the root of hybrid D, the observations made do not elucidate the role of photosynthate in increasing nitrate uptake. There was no genetic difference in partitioning of nitrate (per cent of total) among the plant parts; however, the hybrids differed in amounts of nitrate stored in stalks and midribs. Hybrids D and B accumulated more nitrate than A and C.Although two of the hybrids (A and C) with highest nitrate reductase activity had the lowest concentrations of nitrate in all plant parts, nitrate reductase activity was not correlated with accumulation of nitrate or reduced N for the four hybrids. Uptake and flux of nitrate were not numerically related to accumulation of reduced N for the four hybrids. Among the four hybrids, nitrate flux was not associated with level of leaf nitrate reductase activity. None of the individual parameters, as measured, would serve as an index for reduced N accumulation for these four hybrids. When the hybrid pairs were compared separately, it was evident that both rate of nitrate flux and level of nitrate reductase activity affect the accumulation of reduced N by the plant.Relative to the other hybrids, hybrid D that accumulated the most reduced N and nitrate as a 23-day-old seedling had the least reduced N in grain plus stover at maturity under field conditions. Hybrid C that had high nitrate reductase activity as a seedling had low nitrate reductase activity after anthesis under field conditions. These changes in metabolic activities with plant development and different environments illustrate the problems encountered in attempting to develop simple physiological or biochemical screening criteria useful in identifying superior cultivars at the seedling stage.

Relationship between Nitrate Uptake, Flux, and Reduction and the Accumulation of Reduced Nitrogen in Maize (Zea mays L.): II. EFFECT OF NUTRIENT NITRATE CONCENTRATION.

Two maize hybrids were grown under growth chamber conditions on solution or vermiculite medium that contained 2.5, 7.5, or 15 millimolar nitrate. The objectives were to determine: (a) the effect of nitrate supply on N metabolism and growth and (b) the interrelationship between nitrate uptake, flux, and reduction on the accumulation of reduced N and nitrate by the various plant parts and for the whole plant.Increases in nutrient nitrate concentration caused increases in (a) shoot, but not root, dry weight and (b) nitrate uptake, flux, and reduction and accumulation of nitrate and reduced N by the aerial plant parts of both hybrids. Increases in nitrate supply resulted in decreases in nitrate reductase activity and negligible increases in reduced N in the roots of both hybrids. At 2.5 and 15 millimolar, but not at 7.5 millimolar, external nitrate, hybrid B had higher rates of nitrate uptake and flux. Hybrid B also had lower nitrate reductase activity at all levels of external nitrate and accumulated less reduced N than did hybrid C, except when the plants were grown at 2.5 millimolar nitrate. Correlation values between nitrate uptake and flux were significant for each hybrid and for both hybrids, whereas correlations between nitrate reductase activity and flux and nitrate reductase activity and uptake were significant only for a given hybrid. The correlation values (pooling of all data) between (a) nitrate uptake, flux, or reduction and the accumulation of reduced N by the whole plant, and (b) flux x nitrate reductase activity and accumulation of reduced N were all positive and statistically significant. Because nitrate uptake, flux, and reduction (as measured) were all closely associated with the accumulation of reduced N by the plant, all three parameters should be measured in attempts to estimate the genetic potential of a maize genotype to accumulate reduced N.

Grain Protein Accumulation and the Relationship between Leaf Nitrate Reductase and Protease Activities during Grain Development in Maize (Zea mays L.): I. VARIATION BETWEEN GENOTYPES.

Four maize hybrids, two with high and two with low levels of postanthesis nitrate reductase activity were grown under field conditions. The characteristic enzyme patterns had been established in previous work. Nitrate reductase and proteases were measured in three representative leaves (ear leaf, fourth leaf above and fourth leaf below the ear) at intervals throughout the period of grain development. Concurrent with enzyme sampling, other plants were harvested and subdivided into top, middle and lower leaves, husks, stalks, and ear. Dry weights, nitrate, and reduced N were determined on all plant parts for each sampling. These data established the rate of N accumulation by the grain and depletion from the vegetative material and provide some insight into the relation between newly reduced and remobilized N and accumulation of grain N. Other plants were harvested at maturity for yield and harvest indices for dry weight and N.Nitrate reductase activity was higher in comparable leaves from the high than from the low nitrate reductase genotypes throughout the grain development period. There was no mathematical correlation between nitrate reductase activity and nitrate content of the leaves or stalks, however the high nitrate reductase genotypes maintained a higher amount of nitrate per plant (largely in the stalk) during the later stages of grain development. From the patterns of plant nitrate content it was deduced that the low nitrate reductase genotypes terminated nitrate absorption sooner than the high nitrate reductase types. Proteolytic activities (casein as substrate at pH 5.5 and 7.5) were higher and increased earlier in the low than in the high nitrate reductase genotypes. The "low nitrate reductase-high protease" genotypes had a higher percentage of grain N, and higher harvest index for N than did the "high nitrate reductase-low protease" genotypes. These results permit the tentative conclusions that: (a) redistribution of vegetative N accounted for more of the grain N in the low than in the high nitrate reductase genotypes; and (b) leaf protease activities are more closely related to the accumulation of grain N than leaf nitrate reductase activity.

Some New Aspects of the in Vivo Assay for Nitrate Reductase in Wheat (Triticum aestivum L.) Leaves: I. REEVALUATION OF NITRATE POOL SIZES.

Experiments were carried out to clarify problems encountered in measuring metabolic and storage pool sizes of nitrate in wheat leaf sections with an in vivo nitrate reductase assay. The leaf sections were from seedlings grown on 15 millimolar nitrate. Data obtained show that the cessation of nitrite accumulation, used as an index of the active nitrate pool size, could be caused by lack of anaerobiosis in the assay system, the lack of energy for nitrate reduction, or a loss of nitrate reductase activity. Availability of nitrate was never the limiting factor in this system. It is concluded that pool sizes of nitrate cannot be determined in wheat leaves with the in vivo assays employed.

Pathway for Nitrate Assimilation in Corn (Zea mays L.) Leaves: Cellular Distribution of Enzymes and Energy Sources for Nitrate Reduction.

The localization of enzymes responsible for nitrate assimilation and the generation of NADH for nitrate reduction were studied in corn (Zea mays L.) leaf blades. The techniques used effectively separated mesophyll and bundle sheath cells as judged by microscopic observations, enzymic assays, chlorophyll a/b ratios and photochemical activities. Nitrate reductase, nitrite reductase, and the nitrate content of leaf blades were localized primarily in the mesophyll cells, although some nitrite reductase was found in the bundle sheath cells. Glutamine synthetase, NAD-malate dehydrogenase, NAD-glyceraldehyde-3-phosphate dehydrogenase, and NADP-glutamate dehydrogenase were found in both types of cells, however, more NADP-glutamate dehydrogenase was found in the bundle sheath cells than in the mesophyll cells. These data indicate that the mesophyll cells are the major site for nitrate assimilation in the leaf blade because they contained an ample supply of nitrate and the enzymes considered essential for the assimilation of nitrate into amino acids. Because the specific activity of nitrate reductase was severalfold lower than the other enzymes involved in nitrate assimilation, nitrate reduction is indicated as the rate-limiting step in situ. A sequence of reactions is proposed for nitrate assimilation in the mesophyll cells of corn leaves as related to the C-4 pathway of photosynthesis.

Patterns of proteolytic enzyme activities in different tissues of germinating corn (Zea mays L.).

Profiles of pH dependence and activities of live proteolytic enzymes, amino- and carboxypeptidase and endopeptidases active at pH 3.8, 5.4 and 7.5, with casein as substrate, were determined in crude extracts from the various organs of corn seedlings during germination and early development (30°C, dark, 8 d). With respect to the endopeptidases, caseolytic activities at pH 3.8, 5.4 and 7.5 in extracts from endosperm increased concurrently with loss of endosperm N during germination; however, the relative amounts of the pH 7.5 activity were very small. In scutellum extracts, caseolytic activities at both pH 5.4 and 7.5 increased during the initial stages of development but only the increase at pH 5.4 was concurrent with loss of scutellar N. In shoot extracts, caseolytic activities at pH 5.4 and 7.5 were very low and remained relatively constant. There was a progressive increase in shoot N with time. In root extracts, caseolytic activities at pH 5.4 and 7.5 were higher (3-fold) than in shoot extracts. The activity at pH 5.4 remained constant while the activity at pH 7.5 increased during germination. The rate of accumulation of N by the root was low after day 5. The pattern and ratio but not the amounts of the pH 5.4 and 7.5 caseolytic activities of the root were similar to those observed in senescing leaves of field-grown corn. Addition of mercaptoethanol increased (several-fold) the caseolytic activities at pH 3.8 and 5.4, especially the latter, but not the pH 7.5 activity in endosperm extracts and increased the pH 5.4 activity in extracts from scutellum (30%) and roots (30%) while the effect in shoot extracts was negligible. Carboxypeptidase activity was relatively low in young tissue (root tip, 3-d-old shoots) and increased with development of the various organs except the roots (whole) where the activity remained relatively constant. The increases in carboxypeptidase activities were concurrent with decreases in N from endosperm and scutellum; this result indicates that this enzyme in these tissues may be involved (cooperatively with endopeptidases) in the mobilization of reserve protein.Of all the enzymes tested, only carboxypeptidase activity was markedly (in excess of 50%) inhibited by phenylmethylsulfonylfluoride. Only aminopeptidase activity was found in appreciable amounts in endosperm and scutellum of dry kernels. Aminopeptidase activity was highest in organs with high metabolic activity (scutella, shoot, root tips) and decreased in plant parts undergoing rapid loss of nitrogen (endosperm, senescing leaves).