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A Xerox Education Company SOMMER, Harry Edward, 1941- INFLUENCE OF 2[4 DICHLOROPHENOXYACETIC ACID ON NITRATE REDUCTASE AND PROTEIN IN WILD CARROT L.) TISSU E CU LTURE.(DAUCUS CAROTA L.) TISSUE CULTURE.(DAUCUS
The Ohio State University, Ph.D., 1972 Botany
University Microfilms, A XEROX Company, Ann Arbor, Michigan
THIS DISSERTATION HAS BpEN MICROFILMED EXACTLY AS RECEIVED. INFLUENCE OF 2,4 DICHLOROPHENOXYACETIC ACID ON NITRATE
REDUCTASE AND PROTEIN IN WILD CARROT fDAUCUS
CAROTA L.) TISSUE CULTURE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Harry E. Sommer, 3,S. in Agriculture, M.S. ★ * * * *
The Ohio State University
1972
Adviser Dept, of Botany PLEASE NOTE:
Some pages may have
indistinct print. Filmed as received.
University Microfilms, A Xerox Education Company ACKNOWLEDGMENTS
I wish to thank the Graduate Committee of the Botany
Department for their support and advice.
I wish to thank the following for their support, ad vice, and aid:
Dr. R. W. Sharp, Dr. C. A. Swanson, Dr. F. E. Deather- age, Dr. J. A. Schmitt, Dr. R. A. Popham, Dr. M. L. Evans,
Dr. David Lee, Dr. Ruy de Araujo Caldas, Dr. Linda S. Caldas,
Dr. Henrique Amorim, Alta Scrimsher, Sheila Long, Jane Etta
Holiday, Sara Hill, Cheryl Sands, Anna Bezruczko, and Roy
Postle.
I wish to thank the Department of Biochemistry, De partment of Microbiology, and AEC for financial support.
Finally, I wish to thank my wife for bringing supper to the laboratory during the longer experiments and her other aids to the completion of this work. VITA
25 July, 19 4 1 ...... Born-Chatham, New York
1963...... B.S. in Agriculture, University of Vermont, Burlington
1966...... M.S., University of Maine, Orono
1966-1968 ...... Officer, U.S. Army
iii TABLE OP CONTENTS
Page
ACKNOWLEDGMENTS...... ii
VITA ...... iii
LIST OF T AB L E S ...... v
LIST OP FIGURES ...... vi
INTRODUCTION ...... I LITERATURE REVIEW ...... 6
MATERIALS AND METHODS ...... 51
RESULTS ...... 60
DISCUSSION ...... 87
SUMMARY ...... 105
BIBLIOGRAPHY ...... 109
iv LIST OF TABLES
Table Page
1. Activity of Nitrate Reductase and Nitrite Disappearance in Various Wild Carrot Preparations ...... 61
2. Ammonium Sulfate Fractionation of Wild Carrot Extract ...... 62
3. Effect of Breakage Buffer Components .... 63
4. .Assay Mixture Components and Nitrate Reductase Activity ...... 65
5. Effect of Molybdate on Assay of Nitrate R e d u c t a s e ...... 6 6
6 . n moles Nitrite/ml Nutrient ...... 76
7. n moles Nitrite/ml Nutrient ...... 8 6
v LIST OP FIGURES
Figure Page
1. Activity of Nitrate Reductase vs Time and vs Amount of Enzyme ...... 67
2. Effects of pH and Substrate Concentration on Nitrate Reductase Activity .... 68
3. Fresh weight/culture ...... 70
4. Protein/culture ...... ; • 71
5. n moles NOg Reduced/min/Culture . , 72
6 . n moles NO^ Redueed/min/mg Protein 73
7. [NO^J in Nutrient 74
8. [P0|] in Nutrient 77
9. [P0=] in Nutrient (High Phosphate Nutrient) 78
10. Fresh Weight/Culture (High Phosphate Nutrient) ...... , 79
11. Protein/Culture (High Phosphate Nutrient) 81
12. n moles NOr Reduced/min/Culture (High Phos phate Nutrient) ...... 82
13. n moles N0^ Reduced/min/mg Protein 83
14. [NO^J in Nutrient (High Phosphate Nutrient) 84
vi INTRODUCTION
The research described in this dissertation is part of
a series of studies on the control of embryogenesis in wild carrot (Daucus carota L.) tissue cultures that were
initiated by Dr. D. K. Dougall while he was at the Ohio
State University. We were concerned primarily with discover
ing the biochemical differences which existed between those
cultures giving rise to embryos and those not doing so.
The wild carrot tissue culture system was first de
scribed by Wetherell and Halperin (1) and later further
grown on a nutrient^- that included ammonium and 2,4 D
(2,4 dichlorophenoxyacetic acid.). When these cultures
were transferred to a nutrient without 2,4 D, embryos formed.
When ammonium was omitted from the nutrient containing
2,4 D. transfer of the culture to a nutrient without 2,4 D
did not result in embryogenesis. Growth also was slow in
the absence of ammonium. Based on these reports, we con
cluded that an investigation of the ammonium requirement
provided a logical point to attack the metabolic processes
connected with embryogenesis. The initial steps of nitrate
utilization and the effects of 2,4 D on this process appeared
l Nutrient and nutrient media are used as synonymous terms in plant tissue culture. Abbreviations conform to those recommended for use in the Journal of Biological Chemistry. 1 to be the most likely avenue of approach.
An initial check of the literature led to a paper by
Beevers et al. (3) on the effects of 2,4 D on nitrate metabolism in corn and cucumber. C o m was chosen as a plant relatively resistant to the phytotoxic effects of
2,4 D, while cucumber, like many dicotyledons, is sensitive to 2,4 D. For corn, an increase in nitrate reductase ac tivity was found for all treatments. In the case of cucum ber, the application of 2,4 0 to the plants resulted in a lower specific activity for nitrate reductase during the period 4-12 hours after spraying. Control levels of nitrate reductase were regained at later sampling times. Since wild carrot is a dicotyledon, we might expect it to show a lower specific activity for nitrate reductase when treated with 2,4 D.
Further checks of the literature were made concerning inorganic nitrogen utilization by embryos in culture. This showed .that in culture embryos differ greatly in their abil- * ity to utilize nitrate (4). Embryos from ripe seeds of oats could utilize either ammonium or nitrate for growth (5).
Rijven (6 ) reported excised torpedo stage embryos of Ama- gallis arvensis L., Arabidopsis thaliana (L.) Heynh., Cap- sella bursa-patoris (L). Moech, and Sisymbrium orientale
L. did not grow with nitrate as a nitrogen source. Amagalli3 arvensis L. did show significant growth with ammoni.um. The only inorganic source of nitrogen that gave significant i growth with the other three species was nitrite. In longer term experiments designed to determine if the em bryos could adapt to*utilize nitrate, no evidence, based on growth, could be obtained for an adaptive response in freshly excised embryos. However, while no nitrate re ductase was detected in freshly excised capsella embryos, nitrate reductase was detected after 2 0 hours incubation of the embryos with nitrate. For quantitative studies of nitrate reductase Rijven used 16 day old wheat embryos.
These embryos showed a tenfold increase in nitrate reduc tase after 24 hours on nitrate supplemented sucrose.
Raghavan and Torrey (7) pursued the problem of nitrogen nutrition of embryos in orchids. The embryo in a mature orchid seed resembles that of the globular stage in di cotyledons. The embryos were grown until plantlets developed.
During the first 14 weeks on nitrate the orchids showed an increase in ng N/mg dry weight of seedlings only slightly exceeding those on nutrient without a nitrogen source. The orchids on nitrite showed even less of an increase than those without nitrogen. However the embryos on ammonium salts in some instances showed almost a doubling of ng N/mg dry weight. Ammonium nitrate supported growth appeared to follow the same pattern as those on other ammonium salts.
The orchids on ammonium salts also showed a greater degree of development than those on nitrate. The superiority of ammonium as a nitrogen source for growth continued until 60 day old plantlets were transferred from ammonium nitrate nutrient to fresh ammonium nitrate or sodium nitrate nu trient. Growth was comparable on both nutrients 6 days after transfer. Determinations of nitrate reductase done on seed lings grown on ammonium nitrate gave negative results. Nega tive results were obtained also for 20 and 40 day old seed lings after transfer to sodium nitrate nutrient, even after
100 days on sodium nitrate. However if 60, 80, and 100 day old seedlings were transferred to sodium nitrate, nitrate reductase was found in their extracts, as might have been expected from the growth results. From the results sum marized above, it is evident that we could expect different patterns of inorganic nitrogen utilization in embryos at different developmental stages.
Based on the above information a hypothesis was con structed that stated 2,4 D prevented the utilization of nitrate by the cultures of Wetherell and Halperin. The mechanism could be a direct inhibition of the synthesis of nitrate reductase or an indirect inhibition by stopping embryogenesis in the cultures of Wetherell and Halperin at a stage prior to the development of competence for the synthesis of nitrate reductase. While other factors could be involved in preventing the utilization of nitrate, it was decided to determine the nitrate reductase levels in ex tracts of the cultures. If the nitrate reductase levels were lower in the presence of 2,4 D, it would help establish the plausibility of both mechanisms.
We should at this point note that the wild carrot tissue culture system of Wetherell and Halperin had been developed and used to study embryogensis in vitro at a morphological level. The primary means of evaluation re ported were histological features or the relative numbers of organized structures formed. When the present investiga tion with the system was started, little information was a- vailable on the nutritional requirements other than nitrogen sources, uniformity of growth, or their response to batch cul ture. The experiments were started using the cultures and a nutrient medium formulation supplied by Dr. D. F. Wetherell, and using general culture procedures that had proved success ful in the past with other tissue cultures. An examination of the system at a biochemical level was planned, necessitat ing caution against the possibility that some part of the system might prove inadequate at the biochemical level. This could result in our attributing effects to one cause, that might really be the result of another. For this reason I de cided to monitor selected constitutents of the nutrient, namely phosphate, nitrate and nitrite. LITERATURE REVIEW
In this review we shall consider first the nature of the enzyme nitrate reductase, then what is known about ni trate metabolism and its regulation in tobacco tissue cul tures, and finally we shall describe in further detail the wild carrot tissue.culture system.
In green plants, the enzyme nitrate reductase catalyzes
the reduction, of nitrate to nitrite. This is the first step
in the anabolism of nitrate by the plant. Several reviews
on nitrate reductase (8,9,10,11,12) have covered not only
the enzyme from plants, but also from other sources. Since
the isolation and characterization of the enzyme was not a
major objective of my research, I intend only to outline
some general properties of the enzyme from green plants.
Nitrate reductase as it is currently obtained and
assayed shows several activities (12). The activities are
in part dependent on the-source of the enzyme. The typical
pattern is a pyridine nucleotide diaphorase, a flavine
nucleotide-nitrate reductase, and a molybdate-nitrate reduc
tase. A crystalline nitrate reductase preparation from
neurospora showed NADPH-nitrate reductase, NADPH-*" cytochrome
^"Abbreviations for cofactors are those used in the Journal of Biological Chemistry. c reductase, FADA-nitrate reductase, and reduced methyl- viologen-nitrate reductase activity (13). Even after disc electrophoresis these activities were in the same protein band. Cytochrome ^ 5 5 7 also coelectrophoresed with the nitrate reductase activity. The nitrate reductase was ac
tivated by phosphate. Green plant nitrate reductases have not been as highly purified or as well characterized
as the assimilatory one from the neurospora.
The first report of a pyridine nucleotide-nitrate re ductase from plants was by Evans and Nason (14). The enzyme was purified 60-fold from young soybean leaves. It could
utilize either NADPH or NADH about equally well. FAD was
required for full activity, while FMN caused only slight
activation. Based on assays with crude homogenates from
roots or leaves of 6 other plants, they claimed that the
other plant nitrate reductases could use either NADH or NADPH.
Beevers et al. (15) further investigated the question of the
pyridine nucleotide specificity of higher plant nitrate
reductases. Of 16 species of plants, extracts from all could
utilize NADH for the reduction of nitrate. The optimum
pH was 7.5. Only 4 plants could utilize NADPH, and of these
only the soybean leaf extract could utilize NADPH to a sig
nificant extent (50%) compared to its ability to utilize
NADH. Further investigation with partially* purified nitrate
reductase showed that if soybean leaves were homogenized in
the absence of cysteine, NADH and NADPH were about equally effective as reductant sources. With either pyridine nuc leotide the optimum pH for nitrate reduction was 6.5* Ni trate reductase could not be obtained from corn without cysteine in the homogenizing media. However with NADH' the^ optimum pH for nitrate reductase was near 7.5, but with
NADPH less activity was obtained and the pH optimum was 6.5.
Paneque and Losada (16) investigated the pyridine nucleo tide activity of an 800 fold purified nitrate reductase from spinach. It was specific for NADH. The oxidation of
NADH was stoichiometric with the formation of nitrite;
NADPH could not act as an electron donor unless FMN and
NADP reductase were added. According to Deckard (17) the
NADPH dependence of nitrate reductase in c o m is controlled by the presence of a phosphatase that converts NADPH to
NADH. Ferguson (18) in his investigations on Spirodela oligorhiza found the nitrate reductase in the crude homo- genate was NADH specific. It did not utilize NADPH and the extract did not inhibit a nitrate reductase that utilized
NADPH, However, with a very similar plant, Lemna minor,
Sims et al. (19) found NADH and NADPH-specific nitrate reductase activities. If L. minor was grown on sucrose,
NADPH specific activity increased over 20 fold. The activ ities were also reported to have different regulatory prop erties. Klepper et al. (20) have recently reported an in vivo assay of nitrate reductase for several plants. After infiltrating the tissues with substrates for several pathways, they concluded nitrate reductase used NADH derived from the oxidation of glyceraldehyde-3-phosphate as its main source of reductant. In their review Beever-s and Hageman .
(1 2 ) concluded that for higher plants nitrate reductase was
NADH-dependent in the initial step. They also concluded that NADPH was utilized indirectly through the action of a diaphorase or a phosphatase that was not part of the nitrate reductase complex.
As mentioned before, FAD was an essential cofactor for soybean nitrate reductase (14), and neurospora nitrate reductase has an FADH-nitrate reductase activity associated with it (13). Spencer (21) found for his NADH specific nitrate reductase from wheat, the addition of FAD resulted in a 3 fold activation, while FMN was ineffective. However,
Paneque et al. (22) were able to carry out the reduction of nitrate with a 130 fold purified nitrate reductase using
FMN reduced with dithionite. Reduced FAD, reduced benzyl or methyl viologen also could be used. Chemically reduced
FMN-and FAD were about equally effective in reducing nitrate reductase. FMN also functioned when added with NADPH and a transhydrogenase. Zumft et al. (23) have investigated the i structural and functional role of FAD in the NADH nitrate reductase from chlorella. After passage through a sephrose column, the NADH nitrate reductase was activated by the ad dition of FAD or FMN. FAD was more effective.. The amount of NADH oxidized was nearly equal to the nitrite produced. PAD also protected NADH nitrate reductase from heat inactiva tion at 45°C for five minutes. FMN provided about 10% pro tection, while NADH, methyl viologenr benzyl viologen or- nitrate provided, none. Further work (24) showed, the FAD pro tected the NADH diaphorase activity. The NADH diaphorase activity was inhibited by 0.1 mM p-chloromecuric benzoate' and in the absence of FAD was inactivated by heating to 45®C for 5 minutes. The flavin nucleotide nitrate reductase is unaffected by these two treatments but is inhibited by cya nide. Wray and Filner (25) found that for barley root preparations the NADH-nitrate reductase and FMNH-Nitrate- reductase have the same sedimentation constants in sucrose- gradients. Stoy (26) reported reduced riboflavin can be used to enzymatically reduce nitrate. FMN reduced by dithionite is now a commonly used reductant in nitrate re ductase assays (22,27,28).
As indicated before, cytochrome b i s found associ ated with neurospora nitrate reductase (13). Garrett and
Nason (29) found that with purified preparations of nitrate reductase NADPH caused a slight reduction of cytochrome b55y
If followed by the addition of FAD, the cytochrome bgg^was completely reduced. The further addition of nitrate resulted in the oxidation of cytochrome . Cyanide in amounts suf ficient to stop the reduction of nitrate did not prevent the reduction of cytochrome bgg^ under these conditions. Ev idence for the participation of a cytochrome in the nitrate 11
reductase from green plants is lacking. It was claimed that 55 Ankistrodesmus braunii 202-7c grown in the presence of Fe,
incorporated this isotope into the nitrate reductase obtained by purification procedure that concluded with preparative gel
electrophoresis (30). Earlier it had been shown that with
Ankistrodesmus braunii 202-7C and Chlorella fuca 211-15 im proved iron nutrition increased the reduction of nitrite but 59 not nitrate (31). With Chlorella fuca cultures fed Fe there was no clear cut evidence for the association of the radio
activity with nitrate reductase (32).
Molybdenum has long been considered a component of
nitrate reductase. For the neurospora enzyme it has been
determined that the valency change of the molybdenum is
from +5 to + 6 (33), and that chemically reduced molybdenum
supplied the enzyme along with nitrate resulted in the en
zymatic production of nitrite (34). Cyanide blocked this
step of nitrate reduction. Mineral nutrition experiments with higher plants have shown that molybdenum was required
for normal growth when nitrate is the sole nitrogen source 99 (35,36,37). Working with leaves of soybean plants fed Mo,
Evans and Hall (38) showed that radioactivity per mg of protein
increased with the specific activity of nitrate reductase
during purification. However, maximum purification was only
100 fold. Nicholas and Nason (39) were able to inactivate ni
trate reductase by dialysis against a solution of cyanide. 12
Activity was stored upon the addition of molybdenum tri— oxide or sodium molybdate. Also nitrate was reduced after they supplied reduced molybdate to nitrate reductase,. If. reduced molybdate was supplied to the molybdenum free en zyme, no reduction of nitrate took place. Again these en zymes were only partially purified. Others (40,41) have grown plants with nitrate but without molybdate and have found that upon infiltration of the leaves with molybdate, ni trate reductase activity increased. In addition more recent 99 experiments utilizing Mo have been done (42) . Spinach plants were grown without molybdate and then transferred to a 99 Mo solution. The nitrate reductase was purified, and sub- 99 jected to electrophoresis. Mo was detected only where nitrate reductase was revealed on the gel. Experiments of a similar nature have been done with chlorella (32). How ever the repression of nitrate reductase was relieved by re- 99 moval from an ammonium containing nutrient and Mo added at that time. The radioactivity was closely associated with ni trate reductase during its purification. After a mild heat treatment of the nitrate reductase, molybdenum reduced with dithionite could be used to reduce nitrate. More detailed studies (43) on the effects of molybdate levels on the ni trate reductase levels showed no effects on the level of
NADH diaphorase activity when ammonium grown cells were transferred to nitrate, but the amount of NADH nitrate re ductase activity was highly dependent on the molybdate level 13 of the nutrient. Tungstate, an analogue of molybdate, does not inhibit the growth of ammonium grown chlorella. How ever, tungstate is highly inhibitory when the cultures are grown on nitrate. FMNH-nitrate reductase levels were greatly depressed. The addition of molybdate to the cul tures at least partially reversed the effects of tungstate.
A requirement for phosphate for maximum enzyme activity in the assay system has been reported for neurospora nitrate reductase (44). Several workers (15,37,45,46) have reported higher activities were obtained with nitrate reductase when assayed in the presence of phosphate. Others (14) have not found an activation by phosphate. Most have avoided the issue by using phosphate buffer.
The induction of nitrate reductase and related prop erties have been reviewed (12,47) and will not be treated further until we consider nitrate reductase in tobacco tissue cultures. However, some points related to induction and the physiology of nitrate assimilation that are often overlooked and I wish to emphasize them here. First are the effects of mineral nutrition other than those of nitrogen and molybdate. Harper and Paulsen (48,49) found that defici encies of phosphorus, potassium, calcium, magnesium, sulfur, iron, zinc, and chloride decreased the specific activity of nitrate reductase in .wheat seedlings. The nitrate content of wheat seedlings was decreased by phosphorus and sulfur 14 deficiencies. Second is the effect of ammonium on nitrate* uptake. Using nitrogen-depleted wheat seedlings Minottl et al . (50) showed that nitrate uptake from ammonium nitrate- solutions was lower than from solutions of other nitrate salts and that the difference became greater with time. The same pattern of uptake responses was seen with seedlings raised on nitrate. It was shown by Minotti et al . (51) that for wheat this effect on nitrate uptake was not an indirect result of the inhibition of nitrate reductase.
Ammonium seemed to have no effect on the ability of wheat seedlings to reduce nitrate.
Filner and his students (28,46,52,53,54) have done the only study of nitrate reductase in a plant tissue cul ture system. The studies were done with Nicotiana tabacum
L. var. Xanthi, cell strain XD, grown in suspension cul ture on a modified White*s nutrient M-ID (46). This is a chemically defined nutrient with a nitrate concentration of
2.5 mM. The nitrate concentration appeared to be the factor limiting the growth of the culture. With increasing nitrate concentrations up to 2.5 mM, the fresh weight of the cul tures increased. With M-ID, only 3% of the nitrogen added was left in the nutrient after 10 days* growth. Casein hydrolysate or an amino acid mixture could replace the nitrate and yield the same fresh weight of XD cells in ten days. If growth was limited by nitrogen, a higher yield was obtained with nitrate than with casein hydrolysate as the nitrogen source. Filner (46) claimed.that if excess nitrogen was added to the nutrient both casein hydrolysate and nitrate gave the same fresh weight as was obtained with
2.5 mM nitrate. However, from the data he presented it ap peared that excess casein hydrolysate resulted in a higher fresh weight yield than that obtained with 2.5 mM nitrate.
Urea or y-aminobutyric acid could also be used as nitrogen sources (53). Attempts to grow the cells with ammonium as the sole nitrogen source were unsuccessful (46).
The nitrate reductase obtained from the XD cells was typical of that from many plants (46). It was specific for NADH, had a pH optimum of 7.5, and was more active in phosphate buffer than in Tris. When DPNH was used as the reducing agent, the activity of the enzyme was not influ enced by FAD (3 X 10“ 5 M), FMN (3 X 10~ 5 M), single amino — 3 —2 acids (10 M), or ammonium sulphate (10 M). Activity was not detectable if extractions were made in the absence of cysteine and was labile at 0°C but stable at -80°C. No reduction of nitrite was detectable under the assay - conditions used. When FMN reduced with dithionite became the standard as a procedure (52,53) the crude extract which had previously been used for the assays was found to contain an inhibitor.
The inhibitor and nitrate reductase could be separated by bringing the extract to 50% saturation with ammonium sul phate, collecting and using the precipitate as the source of nitrate reductase. The amount of nitrate reductase found in the cultures varied with the age of the culture (46). No activity was present in the stationary-phase XD cells used to start the cultures. Maximum specific activity was reached in 3 days while maximum total activity was reached in 5 days. No activity could be detected after 12 days in culture. Nitrate was depleted from the nutrient after 10 days. Thus the presence of nitrate reductase seemed to be dependent on the presence of nitrate in the nutrient. Further studies showed approximately a 6 hour lag after the cultures were started followed by 1 2 hours of linear increase in the activity of nitrate reductase per gram of XD cells. In nitrate free nutrient no nitrate reductase was detected during this time.
If the cells were grown on casein hydrolysate, nitrate reduc tase was found only when nitrate was present in the nutrient.
The higher the concentration of casein hydrolysate, the lower was the activity of the nitrate reductase per gram of cells even when initial nitrate levels were identical.
Apparently nitrate is used only to support growth beyond that obtainable with the casein hydrolysate.
Next, the effects of individual amino acids on nitrate reductase induction and growth were tested. The amino acids were supplied at 10“^ M, an amount too low to be useful as a sole source of nitrogen. Only four amino acids did not reduce the activity of nitrate reductase per g of cells during the first 24 hours in culture, namely arginine, 17 cysteine, isoleucine, and lysine. Eleven other amino acids
(alanine, aspartate, asparagine, glutamate, glycine, histi dine, leucine, methionine, proline, threonine, and valine) reduced the specific activity of nitrate reductase during the first 24 hours by more than 75%. The complete picture was further complicated by the fact that after 1 0 days' growth only the cultures with added arginine or lysine had normal or better fresh weight yield. With cysteine growth was reduced 25%. With the other amino acids listed above growth was depressed 6 8 % or more. When amino acids were added to M-ID in pairs 3 effects were found. Alanine, asparagine, aspartate, glutamate, glycine, methionine, proline, threonine, and valine, when added in pairs lowered the specific activity of the nitrate reductase. The second group of amino acids consisted of cysteine and isoleucine.
If either of these two were paired with any member of the first group except for alanine and methionine the specific activity of the induced nitrate reductase was not lowered.
The third group, consisting of arginine and lysine, prevented the lowering of the specific activity of the induced nitrate reductase when added as part of a pair with any member of the first group of amino acids given above.
Studies of 10 days' growth by the XD cells in the presence of various amino acids paired with glycine were conducted. Glycine alone gave only 1-2% of the.normal growth on M-ID. When glycine was paired with one of a group 18 of 1 0 amino acids (alanine, aspartate, glutamate, leucine, methionine, phenylalanine, proline, serine, threonine, and valine) no significant increase in growth over that obtained with glycine alone was obtained. Moderate growth, when com pared to growth on M-ID nutrient, was obtained when glycine was paired with histidine (23%), tryptophane (37%), tyro sine (49%), and isoleucine (65%). Complete reversal of gly cine's growth inhibitory properties was obtained when it was paired with arginine, cysteine, or lysine. There was little correlation between growth achieved in the presence of an amino acid and growth achieved in the presence of that amino acid and glycine except for the cases where glycine was paired with arginine or lysine and to a lesser extent with cysteine. More complex experiments were also reported.
The XD cells grown in the presence of six amino acids (aspar tate, asparagine, glycine, proline, threonine, and valine) resulted in 2% of growth on M-ID. The addition of isoleucine in addition to the other 6 amino acids resulted in 6 6 % of control growth. When both methionine and isoleucine were added with the 6 above amino acids, only 1 1 % of the control growth was obtained. The effects of these 8 amino acids were partially reversed by adding arginine which yielded
56% of the growth achieved with M-ID nutrient. Finally, the cells were grown in the presence of 17 amino acids (the
18 used in the pairing experiments except for tyrosine).
After 10 days' growth the yield was slightly greater than 19
the control's. When arginine or lysine were omitted, growth was about 10% below that of the control. If both arginine
and lysine were omitted, growth vfas only 2 % that of control
growth.
From these experiments it appears that arginine and
lysine can prevent the inhibitory effects of some other
amino acids on growth and on nitrate reductase induction.
Isoleucine is partially effective, and can prevent growth
inhibition or inhibition of the induction of nitrate re
ductase caused by methionine. It appears that the growth in
hibitory effects of the amino acids could be related to the
inhibition of the induction of nitrate reductase. Heimer
and Filner (28) proposed three possible explanations for
the observed effects of the individual amino acids on growth
and nitrate reductase induction:
(1) Single amino acids inhibit growth by specif ically inhibiting nitrate assimilation. (2) Single amino acids inhibit growth by one mechanism and nitrate assimilation by another independent mech anism. (3) Single amino aqids inhibit growth by some mechanism other than inhibition of nitrate assimilation, but as a result of inhibition of growth, many processes are nonspecifically in- hibited. (28)
Two approaches were used: the first was to ascertain if
the individual amino acids inhibited growth on a nitrogen
source other than nitrate. A lack of inhibition would be
consistent with proposition 1 , while inhibition of gorwth
would be consistent with propositions 2 and 3. The second
approach was to select a cell line resistant to growth 20 inhibition by individual amino acids. An alteration in the regulation of the nitrate assimilation pathway in this cell line would support the first or the second proposal.
The first experiment consisted of growing XD cells on
M-ID nutrient, M-ID + threonine, M-ID + threonine + arginine,
M-ID + arginine, nitrateless M-ID + urea, nitrateless M-ID
+ urea + threonine, nitrateless M-ID + urea + threonine + arginine, nitrateless M-ID + urea + arginine, nitrateless
M-ID + y-aminobutyric acid, nitrateless M-ID + y-amino- i ( butyric acid + threonine, nitrateless M-ID + y-aminobutyric acid + threonine + arginine, and nitrateless M-ID + y-amino- butyric acid + arginine. Threonine and arginine were added at 0.1 mM each, while urea and y-aminobutyric acid were added at 3 mM each. The only nutrient that failed to pro duce a normal yield was M-ID + threonine in which growth was greatly inhibited. Thus the cells could grow in the presence of threonine if a nitrogen source other than nitrate was used. This would tend to support the first proposal, but there is the possibility that growth may be regulated by some other mechanism when nitrogen is supplied in a reduced form rather than as nitrate.
Next they reported on the isolation of an XD line re sistant to growth inhibition by 0.1 mM threonine (XDR-Thr);
The first lines used were isolated from XD cultures treated with a chemical mutagen, growth in 0.05 mM threonine, and finally growth in 0.1 mM threonine until growth equalled that of XD cells on M-ID. Later it was found XDR-Thr lines could be isolated by subjecting XD cells to 0.1 mM threonine and isolating the cells that grew. When XDR-Thr cells were grown on M-ID nutrient for ten serial passages and then transferred to M-ID + 0.1 mM threonine to test their re sponse after each passage, it was found they still could grow in the presence of threonine. For unexplained reasons the second and third passages gave low growth on testing, but tests on the remaining passages gave 75% or better of normal growth yields. For a replicate experiment, all growth yields of XDR-Thr cells in the presence of threonine were nor mal. Thus there is some stability in the XDR-Thr line in respect to its ability to grow in the presence of threonine.
The mechanism of resistance to growth inhibition by threonine for XDR-Thr cells was investigated. The cells were grown in the presence of one of eight amino acids
(arginine, glycine, histidine, leucine, lysine, methionine, threonine, or valine). Severe inhibition of growth still occurred in the presence of glycine or methionine. The growth kinetics of XDR-Thr cells were investigated. Unusual kinetics were observed for stationary phase XDR-Thr cells transferred from M-ID nutrient to M-ID + 0.01 mM threonine.
The lag phase for increase in fresh weight lasted for 8 days instead of 2 days. However, the final fresh weight and the time after lag to achieve it was the same as for XDR-Thr cells on M-ID. For growth in terms of protein there was no lag 22 even though the rate was lower than the rate for XDR-Thr cells transferred from M-ID to M-ID nutrient. The final amount of protein per culture was about equal.
If exclusion of inhibitory amino acids from XDR-Thr cells was part of the resistance mechanism, then growth on nitrateless M-ID + 3g/l casein hydrolysate should be low.
However, 10 days' growth produced the same yield with XD and XDR-Thr cultures. No growth kinetics were given.
To determine if the resistant strain took up or metabo lized threonine in some special manner, L-(^C) threonine 14 was supplied the cultures. After 3 days' growth with C- threonine, the XD and XDR-Thr cultures were harvested.
Ninety per cent of the radioactivity initially in the nutri ent was found in the cells, and nearly all of this was still in threonine. Longer term experiments were run with XDR-Thr cells transferred from M-ID to M-ID +0.1 mM threonine and the endogeneous level of some amino acids determined during growth. During 8 days of lag a high level of threonine accumulated. From these results Heimer and Filner con cluded that the lag was not the result of time necessary for the elimination of threonine.
Since arginine can prevent the growth inhibition by threonine, the arginine content and resistance to canavanine of the two strains were compared. Preliminary results showed no appreciable difference in arginine levels between XD and
XDR-Thr cells. However, XDR-Thr cells grown on M-ID + 23 threonine tolerate 5 times more canavanine before growth is inhibited than do XD cells on M-ID nutrient. Free arginine increased about 2.5 fold during lag phase of the XDR-Thr cells transferred from M-ID to M-ID + threonine. However, since we do not know the threshold level of arginine needed to prevent growth inhibition by threonine, it is difficult to determine if this change is of any importance in relation . to a mechanism of resistance.
The ability of the XDR-Thr cells to assimilate nitro gen was evaluated. The O.lmM threonine supplied was in sufficient to support the growth observed and, from previ ous results , is not extensively metabolized. During the 8 day lag-phase for XDR-Thr cells transferred from M-ID to
M-ID+Thr nutrient, nitrate accumulated, nitrate reduc- 1 tase activity developed, and protein synthesized. The
XD cells in M-ID+threonine did not perform any of these functions. When stationary phase XD or XDR-Thr cells were transferred from M-ID to M-ID or M-ID+casein hydrolysate nutrient, there was a 6 hour lag before nitrate accumula tion started. During this lag the internal concentration of nitrate did not exceed the concentration in the nutrient.
This was followed by a period of relatively rapid accumu lation of nitrate. For XD cells in M-ID, accumulation continued for at least 28 hours • reaching about 18 umoles nitrate per gram fresh weight. However in the presence of casein hydrolysate nitrate accumulation by XD cells lasted 24 for about 8 hours after inoculation and leveled off at about
2 jj moles per gram fresh weight. For XDR-Thr cells nitrate accumulation in the presencae or absence of casein hydroly- sate procee~ded more rapidlj than for XD cells in M-ID.
However, in the presence of 0.1 g/1 casein hydrolysate at
14 hours after inoculation, nitrate levels reached about 12
moles per gram fresh weight and remained at that level for the duration of the experiment. At the same time the rate of accumulation of nitrate by XDR-Thr cells in M-ID did slow down, but a nitrate level of 30 nmoles per gram fresh weight was reached by the end of the experiment. A study of the effect of various levels of casein hydrolysate on ni- trate reductase level and nitrate levels in the cells re vealed differences in responses between the XD and XDR-Thr cells. While a given concentration of casein hydrolysate inhibited the induction of nitrate reductase in both strains to about an equal extent on a percentage basis, the per cent decrease in nitrate content was much smaller for the
XDR-Thr cells than for the XD cells. This called for fur- ther investigation.
Since nitrate uptake and utilization were taking place at the same, time in these experiments, an even clearer pic ture of nitrate uptake should be obtained if nitrate accumu
lation in the absence of nitrate reductase could be measured.
Earlier, Heimer, Wray, arid Filner (52) had studied the ef
fects of tungstate on nitrate assimilation in XD cells. The addition of tungstate at 0.05 or 0.1 mM to M-ID nutri ent and growth of cells on this nutrient for 24 hours, re sulted in cells whose extract appeared to contain no ni trate reductase. The umples of nitrate per gram of tissue decreased about 10% over the range 0.01 mM and 0.1 mM tung state. Apparently the tungstate did not have a large effect on nitrate uptake. The addition of O.lmM molybdate 24 hours after the tungstate reversed the effects of 0.1 mM tungstate on the nitrate reductase activity of the extract of both XD and XDR-Thr cells. Cycloheximide, sufficient to
*1 A decrease the incorporation of C-arginine into protein by
98%, added 2 hours before the addition of molybdate, did not prevent the activation of nitrate reductase in the tung state grown cultures. The nitrite reductase level induced by nitrate is not affected by tungstate sufficient to lower detectable nitrate reductase by 95%. Based on this work and further work with barley (25), it appeared tungstate could prevent the activation of the apoenzyme of nitrate reductase.
Thus the experiments on the effects of casein hydroly sate on nitrate accumulation were repeated with 0.02 mM tungstate in the nutrient. The tungstate was sufficient to inhibit the nitrate reductase by 90%. In M-ID nutrient the
XD cells accumulated slightly less (about 16%) nitrate than the XDR-Thr cells. With 0.1 g/1 and 0.3 g/1 of casein hydrolysate the amount of nitrate accumulated dropped for 26 both strains, but far more drastically for the XD cells.
With 0.3 g/1 casein hydrolysate the level of accumulation for
XD cells is 12.5% and for XDR-Thr cells is 52% of that in M-ID nutrient. No kinetics of nitrate accumulation in the pres ence of tungstate were presented. The difference between the 2 strains appeared to lie in their ability to take up and/or accumulate nitrate in the presence of inhibitory amino acids.
Shortly thereafter Heimer and Filner (53) gave a more detailed report on the nitrate uptake system and its regu lation. They tested the effects of tungstate on the rate
1C of N-nitrate reduction and incorporation into protein.
XD cells were grown in nitrateless M-ID + 3m/M urea for 5 days, harvested, and transferred to M-ID nutrient with a 10 15 atom per cent excess of N-nitrate either with or without tungstate at 0,1 mM. The results indicated that the in- 15 hibition of N into protein was proportional to the de crease in extractable nitrate reductase. Further studies were done with XDR-Thr cells harvested in stationary phase.
The development of nitrate reductase activity was blocked with tungstate, 2 hours later cycloheximide was added, and
2 hours after that molybdate was added. The nitrate re ductase level of cells increased after the addition of the molybdate, even though the amount of cycloheximide added was sufficient to decrease the incorporation of ^C-argin- ine into proten by 97%. However# if cycloheximide was added 27
with the tungstate, no nitrate reductase increase was found upon addition of molybdate. This further established the ability of tungstate to act as an in vivo inhibitor of ni trate reductase by a mechanism other than suppression of its synthesis.
Kinetic studies of nitrate uptake and accumulation were done with stationary phase XD cells transferred to M-ID nutrient. The nitrate content of these cells increased very slowly at first but, after 1 0 hours, had reached a con stant rate of accumulation per gram of fresh weight for the duration of the experiment (24 hours). To make sure that
the kinetics observed were not more indicative of recovery
from nitrogen starvation than initial patterns of nitrate up take, XD cells were grown on urea. Exponentially growing
cells were transferred to M-ID with and without tungstate.
For the first 4 hours the moles of nitrate accumulated /g
fresh weight were the same for both treatments. Over the next 4 hours the tungstate treated cultures accumulated more nitrate per gram fresh weight than the untreated cells. In
a similar experiment the exponentially growing cells were
transferred to M-ID with and without urea and nitrate re
ductase activity and nitrate accumulation followed. No ni
trate reductase or uptake was evident at time zero. For both
treatments there was no lag prior to the increase -in nitrate
reductase activity per gram fresh weight. There was about a
one hour lag in nitrate accumulation. The cells grown on 28
M-ID plus urea showed a somewhat variable enhancement in ni trate reductase activity per gram fresh weight over that achieved without urea.
Again exponentially growing XD cells from M-ID nutrient were transferred in M-ID + 0.01 mM tungstate with various levels of nitrate. The amount of nitrate accumulated between the fourth and sixth hours was determined for each nitrate level. The rate of nitrate accumulation was dependent upon the concentration of nitrate in the nutrient. Using a double reciprocal plot, a "K^ 11 .of 0.4 mM and Vmax of 2 - 5 umoles/ hr/g fresh weight were found. vmax was dependent upon the cell batch used. Potasium cyanide and 2,4 dinitrophenol inhibited the accumulation of nitrate. Casein hydrolysate in the presence of tungstate resulted in lower nitrate re ductase levels them with either alone. For XD cells casein hydrolysate and tungstate resulted in 6 - 8 times lower accumulation of nitrate than that obtained in the presence of tungstate; however, the accumulation was slightly higher than that obtained with casein hydrolysate added alone.
Ammonium ions (0.5 mM) inhibited the accumulation of ni trate but only after 6 - 8 hours, in culture. With casein hydrolysate this inhibition of nitrate accumulation was evident after 2 - 3 hpurs, and with nitrite (0.5 mM) the
inhibition was evident in 2 - 5 hours.
If 48 hour old cultures of XD cells in M-ID nutrient were transferred to nitrateless M-ID or MID + 1.5 g/1 of 29 casein hydrolysate, external nitrate seemed to play a very
important role maintaining the nitrate reductase level.
Levels of nitrate reductase and nitrate were high initially
in the 2 day old cells. Without nitrate in the nutrient, nitrate and nitrate reductase fell off rapidly and in parallel after 2 hours. Nitrate did not appear in the nutrient. The cells in casein hydrolysate maintained a slightly lower nitrate content than the control for 24 hours while the nitrate reductase level dropped immediately and sharply, but after 12 hours the enzyme leveled off at a much reduced value.
So far we have considered the inducible nature of ni trate reductase and some means for the regulation of its
induction. But whether the appearance of nitrate reductase results from the activation of a proenzyme or is the result of de novo synthesis has not been considered. Nitrate re ductase induction was shown to result in de novo synthesis of nitrate reductase by Zielke and Filner (54). XD cells 15 were grown on M-ID with 99 atom percent excess N-nitrate
as the sole nitrogen source. After 10 generations on 15 N-nitrate, the cells were transferred to M-ID nutrient 15 14 with 99 atom percent N-nitrate and 20 pc U- C-arginine
(0.1 mM arginine). After 5 days the cells were harvested,
extracted, the nitrate or reductase partially purified and
applied to cesium chloride, for isopycnic equilibrium cen
trifugation. Next the samples were collected in 1-3 drop fractions for the determination of radioactivity, and of nitrate reductase. The results were plotted on gaussian paper to determine the bouyant density ("p) of the nitrate reductase and labeled proteins. Catalase was used as inter nal standard. Likewise cells were grown on M-ID nutrient
1 j ^ ( N - nitrate nutrient) and labeled with U- H-arginine to obtain the "p of N-nitrate reductase and H-proteins. • In later experiments a mixture of 17 amino acids that previ ously (46) had shown little effect upon the induction of IS nitrate reductase was added to dilute the pools of N-amino — 15 acids. The total difference in p between N-nitrate re- 1 A i e ductase and N-nitrate reductase was 0.92%. When N- 1 A nitrate reductase and N-nitrate reductase were mixed, a
2 0 % increase in the bandwidth at half maximum activity was observed. This increase was equivalent to the sum of the
1C 1 4 N-nitrate reductase and N-nitrate band widths plotted together. Thus it was possible to distinguish between a 15 14 mixture of N-nitrate reductase and N-nitrate reductase 15 and nitrate reductase synthesized from a mixture of N 14 and N-amino acids. 15 14 Stationary phase N, C labeled cells were trans- 14 3 ferred to N, H-arginine M-ID type nutrient with 17 amino acids. There was a 12 - 24 hour lag in net protein syn- 3 thesis and fresh weight increase. However, H-arginine was rapidly incorporated into the protein even during the first
24 hours. The fractional changes in of the newly induced 31
nitrate reductase were determined over a 72 hour period
following transfer to the ^ 4N, nutrient. The ^H-proteins
synthesized during the first 4 hours'had a *p indicative of
15 15 • * N-protein, so N-amino acids were still predominant in the
precusor pool. At times greater than 4 hours p" shifted
T A __ more towards that of N-proteins. The decrease in p of
H-labeled proteins was greater than what could be explained
by dilution by net protein synthesis during the period from
24 to 48 hours after transfer. This effect was attributed 3 to the breakdown of H-labeled protein with less than 50%
■^N and their replacement with ^H-labeled protein contain
ing more 1 4 N. 14C labeled protein (intended to be those — 15 labeled before transfer) initially had a p of N-protein,
which gradually decreased until by 48 hours the "p value
corresponded to that of about 50% ^4 N. This indicated the
older proteins were also turning over and since ^4C radio
activity in soluble proteins was relatively constant for 14 the 72 hours, there must be very efficient recycling of C
in the proteins.
By 4 hours after transfer, nitrate reductase had
4 ^ reached 25% of its maximum activity. The p for nitrate re- — 15 3 ductase was less than p for pure N-protein and for the H
labeled proteins. Zielke and Filner offered 2 possible ex
planations for these differences. Either the nitrate re- 3 ductase turns over more rapidly than the average H-protein 3 or the nitrate reductase and the other H-proteins are 32 synthesized from different amino acid pools with different rates of replacement by amino acids. By 8 hours after transfer, the nitrate reductase per gram of cells was relatively constant. There was no significant net protein synthesis until after 12 hours, however the p1 of nitrate reductase continued to decrease. In addition, the half maximum activity band width of nitrate reductase remained constant so the nitrate reductase was not a mix ture of exclusively ^N-protein and ^N-protein. So ob viously the nitrate reductase was undergoing a fairly rapid turnover even early in induction. 15 14 When exponential phase N, C labeled cells were transferred to noninductive nutrient (nitrateless M-ID +
3 g/ 1 casein hydrolysate) nitrate reductase activity de creased rapidly. The P of nitrate reductase decreased also, indicating that during the decay of activity there still was a turnover of nitrate reductase. When exponential 1 1 A N, C labeled cells were transferred to inducing con ditions, the fresh weight, protein level, and nitrate re- dectase level unexpectedly remained relatively constant.
Still P of the nitrate reductase decreased with time. 14 3 Under both conditions the ‘p of C and H labeled protein decreased in a similar manner.
Wetherell and Halperin (1) first reported obtaining embryos from wild carrot tissue cultures grown on a chemi cally undefined nutrient. Callus obtained from root tissue 33 was transferred to rotating liquid culture and in ten day3
embryos were found. When transferred to agar the embryos
developed into plantlets. Some important features of embryo
development were described. The smallest organized struc
tures, about 0.1 mm in diameter, appeared to exhibit polar
ity, i.e., the long axis of their cells were oriented in
one direction. The development of the shoot apex and true
leaves did not take place in rotating cultures, but did take
place when transferred to agar.
Halperin and Wetherell (55) published a more detailed re port shortly thereafter on the development of the embryos and experimental conditions for producing embryos in the tis sue cultures. They included photographs of groups of ten cells and less and claimed these were early stages in embryo-
genesis. However, they were not certain if the smallest
filaments that resembled embryos developed into globular
embryos. Morphologically abnormal embryos were common.
Nutritional and hormonal influences on erabryogenesis were
studied. The composition of the basal nutrient was not
reported, but it consisted of one of five mineral salts
recipes, one of three vitamin recipes, and 2-3% sucrose.
The undefined nutrient included 10% coconut milk and 2,4 D
at 0.1 - 2,0 mg/1. The defined nutrients replaced coconut
milk with either 2 mg/1 of adenine (Ad nutrient) or 0.2
mg/1 of kinetin (K nutrient). Any nutrient that supported
growth also supported some embryogenesis. The pattern of 34
embryogenesis varied little with the basal nutrient used.
However, embryos which developed in callus grown without
coconut milk or with an inhibitory level of auxin> stopped
development at about 1 mm in length.
The first step to obtaining a usable system to study
embryogenesis was taken with the discovery that embryos
could be produced in the absence of coconut milk. Embryos were seen in callus on defined nutrient (Ad or K) with 0.1 rag 2,4 D/1, after 4 - 6 weeks. If 2,4 D was added at 1.0 mg/ 1 , mature embryos did not develop, only globular embryos.
However, to obtain a good yield of callus from explants,
1.0 mg 2,4 D/1 and 1,0 mg kinetin/1 were needed. With Ad
and K nutrients less success was obtained in callus initia
tion. In trying to further define conditions for vigorous
embryo production> they found1 relatively high levels of
2,4 D favor the production of globular proembryos. While
on high 2,4 D these proembryos continued to grow in size.
When transferred to a nutrient that allows embryo maturation,
the larger proerabryos could produce several proembryos.
The tissue grown on Ad nutrient was a homogeneous paren
chymal type. When transferred to auxinless nutrient, this
tissue readily produced embryos. This was taken to indi
cate embryos could be produced in the absence of other speci
alized cells and tissues.
The use of callus cultures to study the conditions for embryo induction and maturation leaves much to be desired in 35 the evaluation of the directness of the effects due to the bulk of other tissues between the embryos and the nutrient,
A partial solution to this problem was provided by Halperin
(56). He grew callus from explants on Ad nutrient for sub cultures, then dispersed the callus in liquid nutrient and sieved the tissue to separate the groups of cells by size.
At this point he called the preglobular and globular embryo- like structures he obtained "Meristems." This terminology was apparently based on the histological disorganization of preglobular forms, and the apparent radial polarity of the large globular forms. These meristems were pipetted onto agar nutrients to test the effects of nitrogen sources and hormones on postglobular embryo development. Meristems developed into embryos on the basal nutrient of salts, vitamins, and sucrose. Indolacetic acid (IAA) or kinetin at 1 mg/1 were toxic. At 1 mg/1 2,4 D promoted prolifera tion of undifferentiated meristems and globular embryos.
At 0.1 mg/1, IAA or kinetin produced linear embryos, while
2,4 D produced globular embryos. With 2,4 D at 0.01 mg/1,
linear embryos developed. Embryo development was independ ent of adenine concentrations at 0.01 - 1 mg/1. To test — 3 the effects of nitrogen sources a basal nutrient with 10 -4 mg/1 kinetin and 5X 10 mg/1 2,4 D was used. Nitrogen was supplied at 20 mM. Nitrogen supplied as a mixture of ammonium and nitrate, or glutamine, supported embryo . development. Asparagine or a mixture of amino acids 36 patterned after coconut milk was somewhat inhibitory.
In the following year Halperin and Wetherell (2,57) published two more papers on this culture system. The first was the result of a more detailed study of the de— velopment of the embryos. The nutrient used contained only mineral salts, vitamins, sucrose, and 2,4 D. Halperin and
Wetherell pointed out in particular that the total elimina tion of coconut milk was very significant, since "studies on the molecular basis for the regeneration of embryos from cultured cells will be hampered by the use of such complex substances as liquid endosperm." From the examina tion of callus sections it was found that embryos grown in callus did have a suspensor. In addition they reported having grown groups of single cells and having observed the entire developmental sequence from single cell to mature embryo. They described the suspensor found on embryos from suspension cultures as those cells not incorporated into the embryo proper. Previously the size and shape of the preglobular proembryos (meristems) had been observed to be variable. This variation was now attributed to the stage at which polarity was established in the clump. Polarized growth and histological, differentiation were inhibited by
2,4 D at concentrations greater than 0.1 mg/1. Dependent upon the size of the clump at the time polarity was estab lished, globular embryos might have a small suspensor, or a very large suspensor, or several globular embryos might have a common suspensor. They further reported that pre globular proembryos arose from small starch filled cells*
These proembryos were described as either linear or spheri cal form. The spherical form was reported to give rise to one embryo, while the linear form may develop one or more globular embryos. In globular embryos the regions of the protoderm, ground meristera, and procambium were discernible*
They also reported changes in the distribution of the starch grains in the embryos during development. They usually found starch grains in the preglobular proembryo, but the starch disappeared as the globular embryo differentiated, disap pearing first in the region of the procambium. In mature embryos starch grains were often present in the cortical region. How much of the changes were observed by follow ing the development of individual proembryos and embryos, and how much was implied from the various forms that can be found in culture is unclear.
As also had been pointed out earlier (55,56) and again in this paper (57), callus tissue' that-had undergone only a few subcultures, readily produced embryos when used as a source of inoculum for nutrient that supported the matura tion of embryos. However, if a culture that had been sub cultured many times was used for inoculum, no embryos formed.
Various natural extracts were tried, but these did not re store the ability to form embryos to the cultures. Later it was reported (58) that prolonged subculturing resulted 38 in. the development of aneuploidy in the callus, and the in ability to form embryos was attributed to this factor*
The next improvement in the state of the art of embryo production came with the report (2 ) of an ammonium require ment for embryo induction. Using a strain that had been grown in culture for over one year on mineral salts, sucrose, vitamins, 2,4 D and kinetin, the relative effectiveness of various nitrogen sources for growth was tested. This callus appeared capable of indefinite growth on a nutrient with nitrate as the sole nitrogen source. The optimum nitrogen level appeared to be about 60 mM. However, when transferred to a nutrient that allowed embryo maturation, no embryos were found. If instead the callus was grown on a nutrient that included 5 mM ammonium, then transferred to embryo maturation conditions, embryos were found. Microscopic examination of the nitrate and the nitrate plus ammonium grown material revealed groups of cells recognizable as proembryos in nitrate plus ammonium grown material but not in the nitrate grown material.
Then other strains were tested for the ammonium effect.
When induction of callus was. attempted on nutrient with nitrate nitrogen and 0.5 mg 2,4 D/1, growth was very slow.
If ammonium was added, a callus rapidly formed on the ex plant. The ammonium was replacable by 0.5 mg kinetin/1.
The callus was sieved and the 45-75 micron fraction inoculated into liquid culture. The tubes contained a basal nutrient (
39 with 10~3 mg kinetin and 5X10~ 4 mg 2,4 D/1. These concen trations of hormones are low enough to allow the production of mature embryos. Nitrogen was supplied as either 60 mM nitrate or 5 mM ammonium and- 55 mM nitrate. The fraction obtained from the nitrate grown callus failed to produce em bryos in either nutrient, while those grown with ammonium in the nutrient produced mature embryos in either nutrient.
It appeared that ammonium was required for the initiation of embryogenesis in the cultures, but not for maturation of the proembryos. However, there is the possibility that conditioning of the nutrient may have supplied reduced nitrogen for the latter stages of embryogenesis.
Halperin (58) published on alternative morphogenetic responses in wild carrot suspension cultures. For a nu trient the salts of Lin and Staba were used, except the nitrogen was as stated for the specific experiment, and the iron EDTA was one half the concentration used by Murashige and Skoog, sucrose was 20 g/1, thiamin was used at 3 mg/1, nicotinic acid at 5.0 mg/1, and 2,4 D varied from 0.1 to
1.0 mg/1. If no 2,4 D or less than 0.01 mg/1 of 2,4D was added, vitamins were omitted. Inoculum for the experiments was obtained from sieved callus or suspensions. The less than 45 micron fraction contained 95% single cells and the remainder were 2 - 5 cell clumps. The 45 - 75 micron frac tion contained free cells and proembryonic masses (previ ously called spherical preglobular embryos or meristems). 40
These fractions were inoculated into rotating liquid cul tures to test their morphogenetic potential.
Callus grown on basal nutrient with nitrate produced a variety of morphogenetic responses in low auxin nutrient.
Some cultures produced a few abnormal embryos, in others death of the culture occurred, and in others disorganized cell clumps developed. After several weeks the clumps gave rise to roots. The clumps contained an area in which there was a central tracheid-like group of cells surrounded by a ring of cambium-like cells. The roots were initiated deep within the cell mass, but exactly where was not established.
Attempts to rigorously define the conditions for rhizo- genesis were unsuccessful.
Proembryos from callus transferred to high auxin liquid nutrient did not continue development beyond the early globular stage but developed into proembryonic masses.
These masses when transferred into low auxin nutrient could produce one or more mature embryos. When the less-than-45 micron fraction is inoculated into low or no auxin nutrient, it yields embryos in numbers approximately equal to the number of multicellular bodies present in the inoculum.
From this it has been concluded that the embryos are not derived from the single cells present in the inoculum. In the case of globular proembryos from suspension cultures, often the protoderm was absent due to the tendency for the outer cell layer to be shed. Premature vacuolation of the cortical cell in the globular and later stages was often ob served. Delayed formation of cotyledonary primordia was common. These observations may seem to disagree in some respects with earlier ones, however we should remember some of those were made with cultures grown on agar.
In the next paper (59) some additional physiologically important points were made. The development of embryos from the less than 45 micron fraction from suspension cultures upon transfer to suspension cultures is dependent upon the density of the inoculum. This holds for the number of embryos that mature and the extent of maturity in ten days.
With fewer cells and clumps in the inoculum, the extent of maturity decreased progressively. However, a certain lower limit had to be reached before the number of embryos pro duced by the inoculum dropped below the number predicted on the basis of more dense inocula. Microculture studies indicated that the multicellular units in the innoculum give rise to embryos. This was achieved in the absence of single cells. Thus apparently a certain number of single cells were not needed to nourish the development of proembryos.
Halperin suggested the effects observed with the more dilute inoculums might result from an excessive leaching of nutrients from the cells in comparison to their rate of replacement. In addition he pointed out that more atten tion should be paid to the effects of conditioning of the nutrient on growth and differentiation’. Halperin and Jensen (60) studied the ultrastructural changes that occurred in the cultures during growth and dif ferentiation. First the large clumps were considered.
Typically they had an inner region of large, highly vacuo lated, thick walled cells. The outer cells were mostly small, thin walled, contained small starch grains, and were densely cytoplasmic. The fate of these clumps depended upon the presence or absence of 2,4 D in the nutrient. In suspension culture in the presence of 2,4 D, these clumps fragmented, then, these smaller clumps grew and underwent fragmentation.‘ The fragmentation seemed to result from a . separation of the cells in the inner region of the clump.
If the clumps were sieved and transferred to auxinless nutrient, then the rate of cell division was accelerated and globular prembryos were formed.
Several observations on the state of some of the cell organelles were reported. As juvenile cells enlarged and became vacuolated, the amount of rough endoplasmic reticulum increased. The free ribosomal content of the cells decreased with age, and in senescent cells there was a marked loss of total endoplasmic reticulum. In auxin-grown cells the ribo somes on the endoplasmic reticulum were always in various ornate polysomal arrangements. The dictyosomes exhibited considerable secretory activity at all stages of cell de velopment. The multivesicular bodies produced appeared to move outward, fuse with the cell membrane, and release 43
their contents into the wall space. There was some question whether all the multivesicular vesicles originated from the
dictyosomes. Mitochondria did not appear to change with cell
age. The plastids contained from one to many starch grains
in the small peripheral cells, but fewer and larger grains
in the vacuolated cells. Lamella do not appear in the
plastids until auxin was removed and embryogenesis commenced.
Additional aspects of the separation of the cells of
clumps and changes in cell structure were also reported.
During the early stages of cell enlargement, membrane-bound
vesicles appeared in the middle lamella. This process con
tinued until intercellular wall areas were replaced by ves
icles. These vesicles resembled the vesicular contents of
the multivesicular bodies. In addition an extensive growth
of fibrillar wall material occurred. The wall material
washed off into the nutrient in great quantities. When the
clumps fragmented, the fragments were polarized with large
vacuolated cells at one end and smaller meristematic cells
at the other. ‘ The number of embryos derived from one clump
was apparently a function of the number of meristematic areas.
Several embryos could arise from one. clump. Some of the
embryos were abnormal. Fragments small enough to pass a
45 micron sieve usually gave rise to single embryos.
Changes in ultrastructure during embryogenesis during
10 days in a 2,4 D-less nutrient were described. The rate
of cell division increased with an increase in cell number 44 and a decrease in cell size with the formation of dense glob ular masses or proembryos. Within 2 days the polysomal con figurations had largely disappeared and smooth endoplasmic * reticulum increased. By 6 days there was a tremendous in crease in free ribosomes in dividing cells and polysomes had disappeared. Nondividing cells had considerable endo plasmic reticulum while only short endoplasmic reticulum profiles were found in dividing cells. The plastids were starch free and lamellae had started to form. Golgi secre tions were reduced and there were fewer dictyosomes in the embryo cells. Microtubules appeared in all cells and were oriented parallel to the cell walls. There was a re duction in tissue separation.
Since the clumps more certainly appeared to be the source of the embryos in suspension cultures, Halperin con cluded. that induction for embryogenesis probably occurred during growth of the explant. What was accomplished in the suspension culture was the reproduction of the induced material (proembryos) by the process of fragmentation. Thus the suspension culture system did not exhibit true adventive embryogenesis, i.e., the induction of a somatic cell to form an embryo.
Since it was possible that the multivesicular bodies associated-with the plasmalemma and walls could be lyso- somes, the localization of acid phosphatase at an ultra- structural level was undertaken (61). The lead salt method 45 was used with B-glyceraldehyde-phosphate, p-nitrophenyl phos phate, glucose-6 -phosphate, glucose-1 -phosphate, and fruc- tose-1, 6 -diphosphate. Cells were harvested in log phase, and incubated in standard nutrient without phosphate for
8-12 hours to reduce nonspecific precipitation. Growth based on net protein increase was not affected during the first 12 hours in phosphate free nutrient. After fixation for 5 minutes with 3% glutaraldehyde in pH 6 .8 , 0.05 M caco- dylate buffer, the tissues incubated with substrate for one half hour at 37°C. Controls consisted of fixed tissue heated -2 for 5 minutes at 100°C, fixed tissue preincubated with 10 M sodium floride, and fixed tissue incubated in a substrate free reaction mixture. Then the tissues were processed for light and electron microscopic observation. B-Glyceral- dehyde phosphate, glucose-6 -phosphate, glucose-l-phosphate, and p-nitrophenyl phosphate were all hydrolyzed enzymati cally at the same cellular sites. In living cells, phospha^ tase, as indicated by lead precipitation, occurred in walls, vacuoles, and occasionally in nuclei and golgi apparatuses.
In dead cells phosphatase activity was extensive throughout the cell, as well as at the sites listed above. In auxin nu trient, distribution of phosphatase changed with age. With numerous cells present, lead precipitation was largely con fined to the comers where intercellular spaces would be formed. As cells aged, the activity extended the length of the middle lamella region. Activity was absent or 46
insignificant in the wall immediately adjacent to the plas- malemma. Acid phosphatase.was found in vacuoles with no in
ternal vesicles and in smaller vacuoles which contained a few
internal vesicles. Vesicles present in the degraded areas
of the cell wall did not show acid phosphatase activity
although the cell wall did. Senescent cells or cells next
to dead cells often showed acid phosphatase in dictyosomes or nucleui. Fructose-1,6 -diphosphate gave lead precipi tate in the nucleolus and to a lesser extent in the nucleus.
Halperin (62) has recently set forth some goals and
criteria for the study of embryogenesis in vitro. First he
stated his criteria for an ideal system for the study of
embryogenesis. It should, by the manipulation of a few variables, allow a homogeneous population of cells to grow
equally well under two sets of conditions, but the cells would be competent for embryogenesis under only one. This
should make possible a finer dissection of the physiology
of induction of embryogenic potential. Such a system is
a goal we have not reached. The wild carrot system is close, however. Part of what is needed with any such system is a
standard test nutrient for quantitative evaluation of the
embryogenic material present. For wild carrot Halperin has
standardized the inoculum at 1 0 , 0 0 0 viable cells/ml and
after 10 days determined the number of embryos present. The
standard nutrient used was apparently the same or close to
the one used in the research to be reported here. 4 7 He started to apply these principles by re-evaluating" the effects of hormones on the callus produced by explants and its embryogenic potential. Callus formation by the ex plants required 2,4 D and was promoted by benzyladenine and to a lesser extent by gibberellic acid #3. However whert the calluses were tested, only those grown with auxin as the sole hormone produced a large number of embryos. This was apparently an effect of the hormones on the cells and not the selection and proliferation of a cell type incapable of embryogenesis. This conclusion was based on the observa tion that those cells grown on cytokinin regain their cap acity for embryogenesis when subcultured on nutrient lack ing cytokinin.
Since kinetin was included in the nutrient when the ammonium effect was found, it was obvious this should be reinvestigated. However, it is apparent from the results presented with the nitrogen treatments used, the original conclusions still stand. All treatments included 40 mM nitrate and were supplemented by 10 mM nitrogen as nitrate, ammonium, glutamine, or casein hydrolyzate. Benzyladenine
(0 . 1 mg/ 1 ) stimulated explant growth under all four treat ments,' few or no embryos were produced upon testing. With
2,4 D as the only hormone, upon testing, only the ammonium and casein hydrolyzate treatments produced a large number of embryos.
Halperin also reported additional work on the cytology 48 of the tissues. The approach used was to divide embryo- genesis into two stages. The first was the stimulation of explant cells to divide and the development of embryogenic daughter cells. The second stage commenced with the dele tion or lowering of auxin in the nutrient and the organi zation of globular proembryos. It was noted cultures com posed entirely of embryogenic cells have not contained other specialized cells. Embryogenic cells tended to partition space equally at division during the first stage.
Nonembryogenic cells tended to enlarge irregularly and to show random orientation of new cross walls. During the second stage the peripherial cells of clumps underwent cell division, but the enlarged cells on the interior remained quiescent. This was demonstrated in part with tritiated thymidine. He also presented 2 graphs. One shows cell number vs_ days in culture. It showed that the number of cells in the minus 2,4 D was greater than those with 2,4 D in the nutrient after the fourth day. This coincided with the end of lag phase of increase in cell number. The second graph showed protein vs days in culture. There appeared to be no significant difference between the protein content of the cultures grown with or without 2,4 D. Halperin felt the similarity was purely fortuitous.
Recently some biochemically oriented studies have been conducted (63,64). In the first study (63) the free amino acid composition of the tissues grown with and without auxin 49
was compared. Only the relative distribution of each amino^
acid was determined and not the size of the total amino-
acid pool. On this basis there was essentially no difference
between 12 day old culture grown with auxin and 12 and 23 day
old cultures grown without auxin. Twenty-two amino acids
were found. Citrulline was the only nonprotein amino acid
and no hydroxyproline was reported.
In order to increase the rate of formation and yield
of embryos from dense cultures (8 ul of packed tissue/ml)
in auxinless nutrient Newcomb and Wetherell (64) had devised
a two-wash procedure. The second wash occurred 24-72
hours after the first wash and transfer to auxinless nutrient. -6 This second wash could be replaced by including 5 X 10 M
2,4,6 -trichlorophenoxyacetic acid (2,4,6 T), an antiauxin,
in the nutrient. The undifferentiated tissue was grown
with 2.3 X 10” 6 M 2,4 D. With 2,4,6 T added there was an
improved homogeneity of developmental•stages and conversion
of undifferentiated tissue. The only morphological differ
ence between the no auxin and 2,4, 6 T cultures was ob
served during plantlet development. The 2,4,6 T grown plant-
lets did not develop root hairs. Based on dry weight, the
rate of growth of the 2,4,6 T cultures were slower than for*
.those without. The cultures on 2,4,6 T showed a higher
rate of respiration (n l 0 2 /hr/mg dry weight) than those
without auxin, when measured at day 7 and days 11 - 12 in
culture. For both treatments respiration decreased with age, and the stimulation of oxygen uptake by 2,4 - dinitrophenol increased by a significant amount in the older cultures. MATERIALS AND METHODS
Tissue Culture Procedures
The cultures used for the experiments to be reported here are derived from a stock culture of wild carrot
(Daucus carota L.) strain CZ supplied by Dr. D. F. Wether- ell. The cultures were grown in the dark on a New Bruns wick psycrotherm controlled environment incubator shaker operated at 25°C and 160 - 170 rpm. Usually the cultures were grown in 50 ml of liquid nutrient in 250 ml Erlenmyer flasks. The most consistent results were obtained if the flasks were acid washed. Contamination checks were made either by examining samples with the microscope or by streaking samples on nutrient agar and potato dextrose agar slants.
The nutrient solution used for the growth of cultures, unless indicated otherwise, has the following composition
(D. F. Wetherell, private communication):
Major Salts
KN03 4.0 g/1 (40.0 mM)
NH 4 C1 0.54 g/1 (10.0 mM)
MgS04 *7H20 0.185 g/1 ( 0.8 mM)
CaCl2 0.166 g/ 1 (1.5 mM)
k h 2p ° 4 0.068 g/ 1 ( 0.5 mM)
51 Iron
FeSO 4 *7H20 14.0 mg/ 1 ( 0.050 mM)
Na2EDTA 18.6 mg/ 1 ( 0.050 mM)
Minor Salts
MnSO 4 -H2 0 7.0 mg / 1 (41.0 U M)
ZnSO4 *7H20 4.0 mg / 1 (14.0 U M)
2.4 mg/ 1 (39.0 11M) H3B0 3 0 . 0 1 mg / 1 ( 0.008 VI M) (NH4)6M o 7°24*4H2° KI 0.38 mg/ 1 ( 2.29 UM)
cuso4 0 . 0 1 mg / 1 ( 0.062 VI M)
Organics •
Sucrose 2 0 . 0 g/i (58.5 mM)
Thiamine HC1 3.0 mg/ 1 ( 8.89 U M)
2.4. dichloro- phenoxyacetic acid 0.5 mg/ 1 ( 2.26 UM) <2,4D)
The major elements were stored as a 10X concentrate, the chelated iron as a 20OX concentrate, and the minor ele ments as a 1000X concentrate, and kept at 0-4°C until used.
Thiamine was added to the nutrient in the form of a freshly prepared 1 mg/ml solution. For 10 times normal phosphate . % experiments, 0.612 g KH2 P0 4 / 1 were added directly to the nutrient solution.
Following addition of the above ingredients to the nu trient, the pH was adjusted to 5.6 + 0.05 with NaOH or HC1 using a pH meter, the nutrient was adjusted to its final i
53 volume, and dispensed at 50 ml per 250 ml Erlenmyer flask.
The flasks were stoppered with a cotton plug, capped with brown paper, and autoclaved for 17 min at 121°C, 15 psi of
steam.
After- the flasks had cooled, or occasionally after up
to 1 week*s storage at 0-4°C, they were inoculated with 2.0 ml (about 70 mg fresh weight of tissue) of an early sta
tionary phase stock culture of wild carrot tissue.
Prior to transfer of cultures from a 2,4 D containing nutrient to one without 2,4 D, the cultures were washed as
follows (L. S. Caldas, unpublished). The cultures were
pooled and transferred to sterile 50 ml screw top glass
centrifuge tubes, and centrifuged 200 Xg at room tempera
ture for 10 minutes. The old nutrient was removed by aspira
tion. An equal volume of fresh nutrient without 2,4 D was
added, the cultures shaken, and allowed to stand for 2 0 -
30 minutes. The tubes were again centrifuged for 10 minute^
as before, and the nutrient removed. This washing pro
cedure was repeated 2 more times. Then an equal volume of
fresh nutrient without 2,4 D was added, the contents of the
tubes pooled, and used to inoculate the flasks in the ex
periments .
Harvest Procedure
The contents of the culture flasks to be harvested were
pooled and the tissue collected by filtration on a milk
filter pad cut to fit the Buchner funnel used. A water 54
aspirator was used for the source of vacuum. After the first
filtration the filtrate was used to resuspend any tissue
that had been carried through with it and resuspend any tissue left behind in the culture flask, and then filtered
again. After the second filtration, the nutrient samples were taken. Finally, the tissue was-washed with 50-100 ml
of cold pH 7.5, 0.1M potassium phosphate buffer. Fresh weights are based on this washed tissue.
Enzyme Extraction Procedure
All extraction procedures were carried out at 0 -4°C.
The washed tissue was transferred to a chilled glass grind
ing vessel and 2 volumes of breakage buffer added. A lower
limit of 2.5 ml of breakage buffer was used. In some in
stances the tissue was stored frozen in the breakage buffer.
The breakage buffer was designed after some found in the
literature (15,45,65,66,67) and refined in work to be re
ported here. The final breakage buffer composition was 0.1
M in pH 7.5 potassium phosphate buffer, 0.4 M in sucrose,
1 mM in EDTA, 1 mM in cysteine, and 1 mM in sodium metabi
sulphite. Breakage was accomplished by 100 full strokes
with a motor driven teflon-glass homogenizer. The homo-
genate was placed in a centrifuge tube and centrifuged at
20,000 Xg for 15 min. The supernatant was removed and the
pellet resuspended in a volume of breakage buffer equal
to the weight of tissue used. A lower limit of 2 ml was
used. Prior to addition to the centrifuge tubes, this 55 breakage buffer was used to wash out the homogenizer tube.
The resuspended homogenate was centrifuged 25,000 Xg for 30 min. The supernatant was combined with the first super natant and the volume measured. A part of the combined supernatants were made 50% saturated with ammonium sulfate, by the addition of a saturated solution ammonium sulfate adjusted to pH of 7.5. The ammonium sulfate treated solu tion was centrifuged at 25,000 Xg for 1 hour. The super natant was removed and the pellet dissolved in a minimum volume (0.3-1.0 ml) of 0.1M pH 7.5 potassium phosphate buffer.
This solution was then applied to a 10 mm X 400 mm G-50
Sephadex column in 0.1M, pH 7.5 potassium phosphate buffer.
The protein fraction was eluted from the column with 0.1M, pH 7.5 potassium phosphate buffer. This fraction contained the major- portion of the nitrate reductase activity.
Nitrate Reductase Assay Procedure
The nitrate reductase assay procedure used was a modi fication of the method of Paneque, del Campo, Ramirez, and
Losada (22,27). The reaction mixture consisted of .0.5 ml of
0.1 M potassium phosphate buffer (pH 7.5), 0.1 ml of 0.1 M potassium nitrate, 0.1 ml of 2 mM FMN, 0.1 ml of 46 mM
sodium dithionite in either 0.1 M, pH 7.5 potassium phos phate buffer, or 0.095 potassium bicarbonate, and double distilled water and enzyme preparation for a total of one ml. Usually the reaction was started by the addition of the
enzyme, and allowed to proceed for 20 minutes at 25°C. The 56
reaction was stopped by vigorous agitation with a vortex mixer. The nitrite produced was determined as wilL be de- » scribed under nitrite determination. To check for nitrite,
destruction, 0.1 ml of 1 mM potassium nitrite was substituted,
for the potassium nitrate in the assay system.
Nitrite Determination
Nitrite was determined by the method of Ferrari and
Varner (27). One ml samples were used. To each sample was added 1 ml of sulfanilamide solution, followed in less
than 10 minutes by 1 ml of N-l-napthylethylenediamine solu
tion. After at least 20 minutes the absorbancy was read at
540 nm. The sulfanilamide solution consisted of:
1 g sulfanilamide (p-aminobenzene sulfonamide)
dissolved in 3N HC1 for a final volume of
1 0 0 ml The N-l-napthylethylenediamine solution consisted of: 20 mg N-l-napthylethylenediamine dihydrochloride
dissolved in 1 0 0 ml of double distilled water
None of the ingredients of the nutrient at the concen
tration used interferred with the determination.
Protein Determination
Protein determinations, were carried out' on 0.1 ml sam
ples. The proteins were precipitated by placing the sample in
2.5 ml of 10% trichloroacetic acid and placing them at 0-4°C
at least overnight. These tubes then were centrifuged at 900- 57
1000 G for 30 min, the trichloroacetic acid decanted, and set to drain. Then the pellets were resuspended in 2.5 ml
absolute ethanol and centrifuged as before, the ethanol was f* then decanted and the tubes drained and the pellets resus pended again in 2.5 ml absolute ethanol. Following centri
fugation, decantation of the ethanol and draining, 1 ml of water was added to each sample tube followed by 1.5 ml
of 5.6% sodium carbonate and 0.5 ml of micro biuret reagent
(K. Caldas, unpublished). The micro-biuret reagent (6 8 ) was prepared by slowly mixing one volume of 1% CuSo^-5 1^0 with
4 parts of 30% sodium hydroxide. The proteins were allowed
to dissolve, and the absorbancy of the solution was read at
310 nm. For some determinations the absorbancy was also
read at 550 nm. Comparison of the results on the identical
samples showed the amount of protein calculated for each
was approximately the same, but the reading at 310 nm gave
greater sensitivity. When the alcohol washes were evapor
ated, the residue did not give a positive biuret reaction.
Standards were prepared from bovine serum albumin, but
were not carried through the initial precipitation and alco
hol wash procedure.
Phosphate Determination
Phosphate was determined by the method of Gomori (69)
One ml samples were used. To each sample 1 ml of Gomori's
molybdate solution was added followed by 1 ml of Elon's de
veloping solution after at least a 1 min wait. Double 58 distilled water was used as a dilutant if needed. After
2 0 min, but before one hour had passed, the absorbancy was
read at 660 nm. Gomori's molybdate solution consists of:
2 .5g Na 2 Mo 0 4 *2 H2 0
25.0 ml ION H2 S04
Double distilled water to a final volume
of 100 ml
The Elon's developing solution consists of:
1 g Elon's developer (mono-methyl para-
aminophenol sulfate)
3 g NaHS0 4
double distilled water to a final volume
of 1 0 0 ml
None of the ingredients of the nutrient at the concen
tration used interferred with the determination.
Nitrate Determination
Nitrate was determined by the method of Lambert and
DuBois (70). A 1 ml sample was diluted to 100 ml. A
10 ml aliquot was placed in a 125 ml Erlenmeyer flask and
15 ml of mixed acid solution was added. One gram of the
color developing reagent was then added. The absorbancy
was measured at 527.5 nm after one-half hour.
The mixed acid solution consists of:
30.0 g citric acid 59
125.0 g ammonium chloride
2 . 0 g dibasic sodium phosphate
0 .1 2 g copper (1 1 ) sulfate
15.0 ml .acetic acid' dissolved in water to a final volume of 750 ml
The color developing reagent consists of:
15.0 g ammonium chloride
1 0 . 0 g sodium citrate
3.0 g manganese (11) sulfate monohydrate
0.9 g cadmium powder
0 . 6 g sulfanilic acid
0.3 g o-napthylamine
mixed and ground together. I
RESULTS
Initial attempts to demonstrate nitrate reductase in wild carrot suspension cultures gave erratic results with either NADH or FMNH as the reductant source. The problem was t narrowed down until it appeared to lie primarily with the use of crude extracts for the assay of nitrate reductase. Crude extracts had been used for many of the studies reported in the literature (12). Several means of fractionating the crude extract were tried. Assays were run for both FMNH* nitrate reductase and nitrite disappearance. The results are shown in Table I. An ammonium sulphate fractionation offered promise of success. The ammonium sulphate procedure was followed up in more detail. Table 2 gives the results and shows that all nitrate reductase present was precipi tated by 50% saturation with ammonium sulphate. Using the
50% ammonium sulphate precipitate, the method for the ex traction of nitrate reductase was checked to verify the de sirability or undesirability of each component (Table 3).
The specific activity of the preparation was increased by the elimination of molybdate from the breakage buffer.
At about this time a large quantity of precipitate from the fractionation of a wild carrot extract with 40% satur ated ammonium sulphate became available as a waste product
60 61
TABLE 1
ACTIVITY OP NITRATE REDUCTASE AND NITRITE DISAPPEARANCE IN VARIOUS WILD CARROT PREPARATIONS
Nitrate Nitrite Preparation Reductase , Disappearance Represented as n moles n moles/minute NO- Produced/min
Crude extract 0.115 4.15
G50 Sephadex Column protein-peak 1.62 0 . 1 2
50% saturation (NH4 )2 S0 4 precipitate 6 . 1 2 0.29 pH 7.5 protamine sulphate supernate 0 8.30
33-40% saturation TABLE 2 AMMONIUM SULFATE FRACTIONATION OF WILD CARROT EXTRACT Precipitated Nitrate reductase Nitrite disappearance From A Asb/20 min A Asb/20 min 0-25%. 0.000 25-33% 0.014 33-40% 0.093 40-46% 0.031 46-50% 0.001 50-57% 0.000 0-50% precipitate 0.094 0.002 50-100% " 0.000 0.005 0-50% supernatant 0.002 0.004 63 TABLE 3 EFFECT OF BREAKAGE BUFFER COMPONENTS Treatment Nitrate Reductase A Asb/20 min/ i mg protein 1 st expt. 2 nd expt. complete ■ • 23.75 26.38 EDTA 21.83 25.62 molybdate 25.09 29.45 sucrose 2 2 . 1 2 23.14 cysteine 23.72 64 from research being conducted by Drs. Lee and Dougall. The preparation was done according to the procedure of Caldas (71)• This fraction contained considerable nitrate re ductase activity. It was found desirable to remove the ammonium sulphate present by passing the redissolved pre cipitate through a G-50 Sephadex column. With this supply of enzyme the assay system was checked. First the essenti ality of each component was tested (Table 4). The slight stimulation by molybdate was checked further, but the re sults did not justify the inclusion of molybdate in the assay mixture (Table 5). Next the assay was checked for linearity with time and the amount of enzyme (Figure 1). Then the assay was checked for pH optimum, and.the effects of varying the concentration of nitrate, FMN, and dithion- ite (Figure 2). These results sustained the validity of the assay procedure adapted from the literature.. . Prelimin ary results showed the nitrate reductase could also utilize NADH, or dithionite reduced FAD or riboflavin, but not NADPH as a reductant source. Both cyanide and phenyl-- mercuric acetate inhibited our FMNH-nitrate reductase. With a nitrate reductase assay and extraction method established, experiments were started to determine the effect of 2,4 D on the nitrate reductase level in wild . carrot tissue cultures. The standard nutrient was. used with or without 2,4 D. The variation between replicate cultures was considerable. These results are presented in 65 TABLE 4 ASSAY MIXTURE COMPONENTS AND NITRATE REDUCTASE ACTIVITY Assay Nitrate reductase A Asb/20 min complete 0.142 enzyme 0 . 0 0 1 boiled enzymes 0 , 0 0 0 buffer 0.123 KN03 0 . 0 0 1 FMN 0.003 dithionite 0.005 -5 complete +10 M Na 2 MoO^ 0.145 - no 3 + no2 - 0.006 66 TABLE.5 EFFECT OF MOLYBDATE ON ASSAY OF NITRATE REDUCTASE Molybdate concentration Nitrate reductase moles/ 1 A Asb/20 min 0 0.128 1 0 - 3 0.119 - »4 1 0 * 0.119 1 0 “ 5 0.124 1 0 - 6 0 . 1 1 1 A A Absorbancy Enzyme of Amount vs. and Time vs. Reductase itrate N of ctivity A 1 Fig. 0.2 0.1 1 2 30 20 10 0 yiue m Exr ct xtra E ml fyjinutes <3 o C\1 < XI XI o E c w) a c >, u 0 .3 .05 .03 .01 peaain 1 preparation a peaain 2 preparation a peaain 3 preparation • 0.1 0.2 a\ Fig. 2 Effects of pH and Substrate Substrate and pH of Effects 2 Fig. Absorbancy / 20min 0.2 0.1 0.1 6.0 Activity. 65 •4 tat (M) te itra N 7.0 ■3 7.5 2 8.0 •1 ocnrto o Nirt Reductase itrate N on Concentration 0.1 j O 2 0 Dithionite(mM) ,2 M ( ) M (p FMN .6 4.6 0.46 2 200 46 2000 69 Tables 6 and 7 and Figures 3-14. From Figure 3 it cam be seen there was little or no difference in the fresh weight of the cultures until after 7 days. The cultures without 2,4 D achieved the greater fresh weight before day 14. From Figure 4 it appears that there is.ho great dif ference in protein content in the 2 treatments until, after the first 7 days. Then the protein content of the cultures with 2,4 D tends to increase, while that of the minus 2,4 D cultures tends to hold steady, and thereafter the protein content of the cultures under both treatments starts to decline. The total nitrate reductase activity showed sharp differences between the two treatments (Figure 5). At 5 days there was little difference between the two treatments. After that time the total nitrate reductase in the cultures with 2,4 D increased until day 9, then fell sharply. The total nitrate reductase of the cultures without 2,4 D dropped sharply from day 5 and appeared to remain at a lower level than the cultures with 2,4 D. The specific activity of nitrate reductase for both treatments started off at approximately the same level and then dropped off.sharply (Figure 6 ). From Filner's (46) results, we might expect that this drop was due to the depletion of nitrate from the nutrient. However, Figure 7 shows there is sufficient nitrate left in the nutrient even after 14 days. The I GRAMS pjg. pjg. 4 2 5 3 6 1 0 3 rs Wih Culure r ltu u /C Weight Fresh a series series a o series series o • series 1, - 2,4 D 2,4 - 1, series • + 2(4D 1, series * 5 2, 2, 2. D .4 -2 +D 2,4 10 S Y A D 15 0 2 7 ° MILLIGRAMS 35 4 Fig. 30 20 25 0 5 rti Culure r ltu u /C Protein 10 S Y A D 15 series 5, +2/4D +2/4D 5, series eis2 + 2,4 D 2, series eis , 2400 -2,4 1, series series series series % 2, + 2,4D - 2,4 D 2,4 - 20 71 72 Fig.5 n Moles NO^" Reduced Culture a series 1, + 2,4 D a series 2,+2,4 D • series \ - 2,4 D o series 2,- 2,4 D 150 125 ■a 75 25 20 DAYS 73 n Moles NO" Reduced^Min/mg Protein a series 1, + 2,4 D a series 1.) + 2,4 D • series 2,- 2,4 D o series 2, - 2,4 D series 1, initial value series 2, initial value 9 8 O) 7 6 5 4 3 2 1 0 5 10 15 20 DAYS m M N0_V 40. 30 50 20 Fig. Fig. 7 0 5 o n : 10 n rent n trie u N in S Y A D a A series 2, + 2,4 +D 2,4 2, series A o series 4, 4, series o o series series o • series series • series 3, + 2,4D 2,4D + 3, series 15 4, 2, - 2,4 D 2,4 - 2,4D - ■+ 2,4 D 2,4 Fig. Fig. 7 74 presence or absence of 2,4 D seems to have little effect on nitrate disappearance from the nutrient. Only a relatively small amount of nitrite was found in the nutrient (Table 6 }. When phosphate in the nutrient was measured, it was found that for cultures both with and without 2,4 D, by day 7 phosphate was essentially depleted from the nutrient (Figure 8 ). This indicates the effects observed might be- due primarily to a phosphate deficiency, rather than the presence or absence of 2,4 D. Recently Nash and Davies (72) have reported that Paul’s Scarlet rose tissue cultures- deplete the phosphate in their MX^ nutrient medium in from 4 to 7 days. They made no attempt to correct this or other deficiencies which they reported in the nutrient medium. We decided to examine the effects of this limitation by phosphate on the wild carrot tissue culture system. We wanted to know if any of the effects observed for 2,4 D were more indicative of the limitation of the culture by phosphate than the effect of 2,4 D. A new series of ex periments were designed with the goal of evaluating the effects of increased phosphate on the cultures. The phos phate level of the nutrient was raised 10 fold. From Fig ure 9, it can be seen that sufficient phosphate remained in nutrient medium after 14 days of culture, and this observa tion indicates that the results were not due to phosphate deficiency. From Figure 10 it can be seen that the cul tures grown without 2,4 D still had the greater fresh weight 76 TABLE 6 n MOLES NITRITE/ml NUTRIENT n moles nitrite/ml nutrient days in + 2,4 D - 2,4 D culture cultures cultures series 2 series 3 series 4 series 2 series 4’ 0 0.5 0.9 0.5 0.5 0.7 5 1 . 8 5.7 7.3 2.4 9.1 7 2.3 7.4 14.4 1.3 9.2 9 5.1 6.5 3.0 2 . 0 2.9 1 2 2.3 3.4 2 . 1 2.5 4.4 14 3.4 1.9 2.9 m m M Fig. 8 Fig. pop] n ti t n utrie N in ] p o [p '-4—; ».. . » ; — 4 s'*- 0 15 10 DAYS a series 4, series a a sre , , D 2,4 - 2, series o ■ series + series 5, +■ D 3, series 2,4 □ • series series • series + series D 2, 2,4 A, - + 24 D 2,4 2,4 D 2,4 2,4 D 77 78 Fig. 9 [pO=Jjn Nutrient High Phosphate Nutrient 7 series 3, + 2,4 D a series 4, + 2,4 D • series 3, - 2,4 D o series 4, - 2,4 D c f DAYS G ram s 10 Fig. 8 10 6 4 2 ' ih hsht Nutrient Phosphate High rs Wih Culure r ltu u /C Weight Fresh S Y A D • series series • o series 4 ^ 2 ,4 0 ,4 2 ^ 4 series o sre 4*,D 4,*2,4D series £ a series 3(+2,4D 3(+2,4D series a 3 - 2,4 0 79 80 at the termination of the experiments. The fresh weight at 14 days was also higher with or without 2,4 D,respectively, than that achieved with the original standard nutrient (cf. Figures 3 and 10). The protein content (Figure 11) of the high phosphate grown cultures with and without 2,4 D in creased up to day 9 with little difference between treat ments. In the following days the protein content of the cultures without 2,4 D increased to the extent of 20% and then dropped slightly, while the protein content of the cultures with 2,4 D remained approximately constant. The maximum protein per culture containing increased phosphate was approximately twice that obtained with the original standard nutrient. The total nitrate reductase activities (Figure 12) were at approximately the same level at day 5. The total activities rose rapidly for 2 to 4 days then fell off. The maximum activity in the high phosphate nutrient was at least twice that with the standard nutrient. The specific activity of nitrate reductase (Figure 13) remained fairly constant from day 5 until day 7 or 9, after which it decreased. The specific activity of nitrate reductase from the cultures with 2,4 D was higher than that from those without 2,4 D at days 5 and 7. Nitrate was not depleted from the nutrient by either treatment (Figure 14). After day 9 more nitrate disappeared from the nutrient of the cultures without 2,4 D than from the cultures with 2,4 D. More nitrate was removed from the nutrient by the cultures Milligrams 70 60 40 50 30 20 10 A ih hsht Nutrient Phosphate High . Protein / C ulture ulture C / Protein . DAYS 0 15 10 X a eis 3, series • 4, series a sre 4, series o eis 3, series + 2,4 D +2,4 D+ 2,4 - 2,4 D 2,4 - - 2,4 D 2,4 - 81 Fig. 12 n Moles NO" R e d u c e d /M in /c u ltu re High Phosphate Nutrient a series 3, + 2,4D & series 4, + 2,4D • series 3, - 2,4D o series 4, - 2,4 D 300 250 200 50 0 5 10 15 DAYS 83 Fig. 13 n Moles NOT Reduced/Min/mg Protein O < High Phosphate Nutrient A series 3, +2,4D a series 4, +2,4D • series 3, - 2,4 D o series 4,-2,4 D 12 ■ series 3, initial value □ series 4, initial value 11 10 c * c 7 2 XJ £U 6 U D TJ (L> 5 CC 4 b " U) 3 a» o 2 2 c 1 0 5 10 15 DAYS 84 Fig. 14 L^°3J n Nutrient High Phosphate Nutrient a series 3, + 2,4 D £ series 4, +2,4 0 e series 3, - 2,4 D o series 4, - 2, 40 50 40 UP 30 20 © \ o 0 5 1 0 15 DAYS 85 grown with the increased phosphate nutrient than those grown with the original standard nutrient. Nitrite in the high phosphate nutrient was higher than in the case of growth on the standard nutrient (Table 7). Some preliminary experiments were done on growing cultures without ammonium. Growth was slow. At best the equivalent of 5 days’ growth on the standard nutrient was obtained in 2 weeks without ammonium. Embryos could be obtained by transferring these cultures to nutrient without 2,4 D. However, the total yield of embryos was low. Perhaps ammonium requirement is not obligatory for embryo production. > 86 TABLE 7 n MOLES NITRITE/ml NUTRIENT HIGH PHOSPHATE NUTRIENT n moles nitrite/ml days in f 2,4 D culture - 2,4 D cultures culture series 3 series 4 series 3 series 4 0 0.9 0.5 0 . 8 0 . 6 5 8.5 14.4 10.9 22.7 7 28.8 50.8 17.9 29.3 9 12.7 18.7 1 1 . 2 15.4 1 2 1.5 3.9 1.7 7.9 14 1.7 2 . 8 1.7 1.7 DISCUSSION The characterization of our nitrate reductase prepara tions indicated that they resembled a typical green plant nitrate reductase (1 0 ,1 1 ,1 2 ) with respect to reductant sources, pH optimum, and other general properties. We were not able to establish a molybdenum requirement since the enzyme denatures during dialysis. This is apparently why the attempts by other investigators to demonstrate a molyb denum requirement have been indirect. After trying many simplified versions of the nitrate reductase assay, we found that the rather laborious procedure described, above was necessary to assure reasonably accurate and reproduc ible results. As cited earlier Heimer and Filner (28) had to adopt a similar method due to the presence of what they called an inhibitor in their extract. More recently Streeter and Bosler (73) have reported results indicating that severe interference with nitrate reductase determina tions also occurred in extracts of soybean leaves. The key step in the assay is the determination of nitrite resulting i from nitrate. A critical study of this aspect of the assay procedure showed that the extracts contained an unidentified substance which seriously interfered with color development. Modification of the commonly used assay procedure (74) was 87 88 necessary. Surprisingly this change revealed a 2- 5-fold increase in the amount of nitrite found. These results now leave a large amount of the literature open to question from the point of view of the soundness of the analytical metho dology used. By developing our own method for the fraction ation of the extract prior to enzyme determination we have improved markedly the accuracy and reproducibility of the nitrate reductase assay. There was a fair amount of variability in the results obtained from identical treatments and this is expected with plant tissue culture systems. Previously workers in this laboratory had been involved with studies on the metabolism of amino acids, particularly arginine, and phenolic compounds in Paul's Scarlet rose tissue cultures. After a period of years the properties of the cultures had become so changed that the last experiments performed with these cultures gave results that could be considered only remotely re lated to those previously obtained either in terms of growth oiT~effects of nutrition on the biosynthesis of the phenolics. This phenomenon is not confined to these cul tures alone. Soybean tissue cultures used for the bio assay of cytokinins frequently have been unusable for this purpose for short periods of time due to irregular growth (J. G. Torrey, personal communication). Likewise Acer pseudoplatanus L. cultures could not be indefinitely 89 maintained as suspension cultures due to diminishing yields but must regularly be transferred to agar nutrient medium or restarted from stocks maintained on an agar nutrient medium {P. Albersheim, personal communication)• Thus the variability found with the wild carrot system was not sur prising but the degree of variation was sufficiently small to permit meaningful interpretation of results. We were successful.in detecting and eliminating, or identifying, several sources of the variability in the wild carrot tissue culture system. For instance, with 5 sets of cultures grown the summer of 1970, the range of fresh weights at 14^15 days for those grown with 2,4 D on the original standard nutrient medium was from 0.65 to 2 . 0 g, while the range in fresh weight for those grown without 2,4 D was from 2.3 to 3.7~g for the same age cul tures. Despite the fact that both ranges were large, they did not overlap. In addition, those sets that showed low growth in the nutrient with 2,4 D also showed low growth in the nutrient without 2,4 D, and those that showed the greater growth did so under both conditions. Therefore the results obtained with a given set of cultures were com parable in terms of the effects of 2,4 D. In part this vari ability was due to differences in the initial inoculum. This source of variation was reduced by not using stock cul tures that showed low growth. Some variability in inoculum size was introduced due to loss of tissue during washing of 90 4 the stock cultures used when setting up experiments on the effects of 2,4 D. Other more easily controlled, but often unexpected, sources of variability were found. Mixing new and used culture flasks in the same experiment resulted in yields grouped in two ranges. After our laboratory moved to new facilities the cultures appeared to be losing their ability to produce embryos in the nutrient without 2>4 D. Inspec tion of the glassware showed rings and spots that had not been seen before. Chromic acid washing of the culture flasks was necessary to alleviate the problem. Redistilled house-distilled water from the Biological Sciences Building proved toxic to the cultures, while redistilled house- demineralized water from the laboratories formerly used had proved a satisfactory water source. The differences in the water supplies apparently was due to the presence of rust inhibitors added to the steam supply and piping in the new laboratory facilities. Street (75) has provided evidence recently for the production of an unidentified gas by older cultures that promoted the initial phase of growth of freshly inoculated cultures. Thus such a common procedure, often necessary for economic reasons, as growing older and freshly inoculated cultures in the same shaker could have influenced the growth of the younger cultures. In our case stock cultures of 91 various ages and experimental cultures were kept on the same shaker. Even when we took all foreseeable and practical precautions, one culture out of 40 might not grow. Wl\ile the variability between identical cultures could in part be controlled, it did mean that the system was not yet re fined to a point where definite conclusions could be based on small differences. The variability in determining protein or enzyme on a per culture basis was due to the necessity of making these determinations on 0.1 ml or smaller samples. It should also be noted that the protein determinations reported here were measured by a microbiuret method wherein inter- ferring substances had been removed by precipitation and washing procedures. This is considered preferable to the more interference-susceptible Lowery method (76). One other point that should be mentioned is that due to failure of the refrigeration system on the shaker, there was a 24 hour period when the cells were, at .40°C, and con siderable amounts of the tissue died. In two weeks the stocks had recovered their normal growth rate. Unfortun ately, this incident took place after the experiments on standard nutrient had been done, but not the ten-fold phosphate nutrient experiments. Standard nutrient growth samples that had been grown as controls for the ten-fold phosphate experiments were lost due to failure of the -20°C 92 cold room; however, fresh weights and phosphate uptake patterns appeared normal. A significant contribution of this research is the discovery that phosphate may be limiting in the wild carrot tissue culture nutrient medium commonly employed. This nutrient has been proposed by Halperin (62) for use as a standard test nutrient for the wild carrot tissue culture system. It seems reasonable that this condition may be another factor contributing to the variability found in these cultures. All stocks were maintained on this stand ard nutrient. It may.seem unreasonable that this deficiency should go undetected for a number of years. However, I have been able to find only 2 papers (72,77) on plant tis sue culture which report changes in phosphate concentra tions in the nutrient during growth. Both involve suspension cultures. As cited before, Nash and Davies (72) reported in 1972 measurements of the phosphate level in the nutrient used for the growth of Paul's Scarlet rose tissue culture. The initial concentration of phosphate was 1 mM, i.e., twice that in the standard wild carrot nutrient medium. With a low inoculum of rose tissue, phosphate in the nutrient was depleted between day 7 and 8 , while with a high inoculum phosphate in the nutrient was depleted between day 4 and 5. They also reported nitrate and sugars were depleted before 14 days. The nutritional changes were in part correlated with changes in the synthesis of protein, RNA, DNA and 93 phenolics. Bellamy and Bielesky (76) earlier.had measured the uptake of phosphate by tobacco tissue cultures in a nutrient containing 2 mM phosphate. By the tenth day phos phate was depleted from the nutrient. However, this report did not lead to investigations of the adequacy of other tissue culture nutrients in terms of what is removed from the nutrient medium. The reason for a lack of concern by most investigators with the quantitative nutritional adequacy of plant tissue culture nutrient media is best understood by quickly review ing the types of criteria used in choosing and evaluating a nutrient medium. Many nutrient media have been used because they supply no more than the minimum requirements needed to support growth and thus whatever phenomena are studied would presumably not be influenced by nonessential components pres ent in the nutrient medium. -These were the first defined nu trient media and were carefully developed in qualitative terms of what was essential. The major salts were usually supplied as dilutions of widely used hydroponic solutions. Most experiments concerned the influence of hormones or vita mins on the cultures with little attention paid to the other components. More recently nutrient media have been chosen . to provide maximum growth. This growth has usually been defined as the amount of increase in fresh weight in 2 weeks to a month. Salts were evaluated in terms of their effect on fresh weight and not determined in the nutrient 94 medium during growth. Both the minimal ingredients and maximum growth nutrient media were developed using one or a relatively few species of plants. Other nutrient media have been chosen because they will support growth of the tissue or show some phenomenon of interest. The most fre quently varied components have been plant hormones or or ganic supplements. The inorganic constituents have been set. and ignored, or two or three well known formulations have been tried and the one giving the best results selected and used. The majority of nutrient media have also been solidi fied with agar. This makes analysis of the nutrient com ponents difficult. The use of agar gel media also probably limited growth to a large extent by diffusion of the com ponents of the medium to and throughout the cultures. Thus, from these considerations, I think there is reason to ques tion the nutritional adequacy of every tissue culture medium in the literature today. This is particularly true when they are used to support the growth of tissues in sus pension culture. For any study of a plant tissue culture system, a primary' criterion essentially ignored to date has been the changes in the nutrient medium during growth. The differences we showed in protein and nitrate reductase levels on the standard and high phosphate nutrients give ample evidence of the problems that can be encountered when changes in the levels of components of the nutrient medium during growth are ignored.- 95 Our results showed that the. differences in fresh weight of the cultures grown with and without 2,4 D were probably due to 2,4 D. The cultures grown without 2,4 D always had the higher fresh weight at the end of an experimental ser ies.. Furthermore, the case of the cultures grown on the high phosphate nutrient medium, the cultures with 2,4 D appeared to have removed less phosphate and nitrate from the nutrient medium than those without 2,4 D. Conversely, the results obtained with low-phosphate medium showed small to zero differences in the uptake of either nitrate or phosphate from the nutrient. For the high phosphate cul- './r » | tures both the difference in fresh weight and in uptake be came evident around day ten. Since most of the fresh weight gained from this point on was the result of water uptake (i.e., net protein synthesis had nearly stopped), the two 2,4 D effects given above may have been related. As Kramer (78) has pointed out, the rapid uptake of water by plants is preceded by active accumulation of salts which decreases the water potential of the cells in respect to their environment 1... resulting in the net entrance of water. Data on the uptake of other ions and sucrose would be needed to completely evaluate this possibility in the carrot cultures. Among the various explanations for the mechanism of action of 2,4 D that appear to fit best here is that 2,4 D may act as an inhibitor of malic dehydrogenase and as. an un coupler of oxidative phosphorylation (79,80). The concentrations of 2,4 D used by Wedding and Black (79,80) were considerably higher than those used here. However, if we assume that the same effects could be exhibited with lesser magnitude at lower concentrations, then in the cul tures with 2,4 D we would expect less energy to be available for active salt uptake. Thus the.cultures with 2,4 D should have a higher water potential (less negative), and absorb less water than the cultures without 2,4 D resulting in the lower weight and lower rate of disappearance of phosphate and nitrate from the nutrient medium. The apparent lack of effect of 2,4 D on phosphate arid nitrate uptake in the case of the original standard nutrient medium might be explained by the inherent variability of tissue culture experiments. However, if the observed differences in weight of the tissue obtained with and without 2,4 D in the original standard nutrient medium are to be explained by differences in the uptake of salts and sucrose by the tissue (78), it would be necessary to reexamine the uptake of other inorganic con stituents of the nutrient medium, such as potassium, or to determine tissue metabolites such as sucrose and malic acid. The lower weight gains obtained on the standard nutrient medium as compared to the ten-fold phosphate nutrient medium could be explained as follows: the phosphate deficiency resulted in a lower rate of oxidative phosphorylation, thus in a lower ability to accumulate salts resulting in less water uptake and less gain in weight. As a consequence 97 there was a masking of -the effects of 2,4 D on salt uptake. The results of Newcomb and Wetherell (64) showing the stimu lation of oxygen uptake by 2,4 dinitrophenol in older cul tures tend to support the assumption that oxidative phos phorylation may have been inhibited by the phosphate de ficiency. The above mechanism of action for 2,4 D could not, however, explain a direct effect of 2,4 D on nitrate re duction by the cultures since in^ vivo experiments (2 0 ) indicated that the source of DPNH for nitrate reduction was not mitochondrial but glycolytic. The direct effects, if any, of 2,4 D on protein syn thesis and nitrate reductase are not clear. The higher maximum nitrate reductase level with the standard nutrient occurred with 2,4 D. The greater initial specific activity of nitrate reductase in cultures on high phosphate occurred with 2,4 D. This might reflect the greater ability of the cultures with 2,4 D to synthesize enzymes due to their higher polyribosome content (60). The most dramatic changes that occurred in the system were found when the phosphate level was increased ten-fold. Fresh weights, protein, and nitrate reductase levels in creased. The changes in fresh weight have, in part, been accounted for above, but these changes also reflect the greater synthetic capacity of the cells as shown by greater net protein synthesis. 98 The original reason for conducting this research was to determine if the reported.requirement for ammonium in the culture nutrient during growth in the presence of 2,4 D in order to achieve embryogenesis after the 2,4 D is removed could be explained by the inhibition of nitrate utilization by 2,4 D. However, the nitrate reductase level and/or specific activity of the cultures grown with 2,4 D equalled or exceeded those from cultures grown without 2,4 D. The above results indicated that the effect of 2,4 D was not due to a decrease in the nitrate reductase levels of the cultures. Calculations based upon the initial content of ammoni um and nitrate in the nutrient medium showed that sufficient * nitrogen was present to produce 44.5 mg of protein per flask from the ammonium nitrogen and 175 mg of protein per flask from the nitrate nitrogen. The amounts of soluble protein (a maximum of 32.5 mg) that were present in the cultures grown under standard conditions could have been derived entirely from ammonium. However, for the high phosphate cultures, the protein (a maximum of 67 mg) must have been in part derived from nitrate since there was in sufficient ammonium present to contribute all the nitrogen needed. We might suspect that due to the phosphate de ficiency the cultures were unable to utilize nitrate and thus needed ammonium as a nitrogen source. However, in the absence of ammonium our preliminary results did not show 99 substantially greater growth by the high phosphate cultures compared to cultures grown, on the standard nutrient. Next, was the nitrate reductase level sufficient to account for the protein in the cultures? By taking the activity of nitrate reductase found, in the cultures and the time through which the enzyme acted, we can, by taking the area under the curves,estimate potential protein synthesis from nitrate. By making such computations we get the fol lowing: For days 5-14 on standard nutrient with 2,4 D the nitrate reductase was capable of reducing nitrate to provide sufficient nitrogen for 115 mg protein. The same calculations for the cultures grown without 2,4 D revealed that only 39 mg of protein could have been produced. In the case of the high phosphate treatments, there was suffici ent nitrate reductase from days 5-14 to have produced re duced nitrogen for 223 mg of protein in the cultures with 2,4 D, and 167 mg of protein in the cultures without 2,4 D. 'Thus, there was sufficient nitrate reductase present to ‘ produce the reduced nitrogen necessary for soluble protein synthesis without considering the ammonium in the nutri ent. • The levels of nitrate reductase (Figures 5 and 11) changed with time as did the uptake of nitrate (Figure 7 and 14). Nitrate disappearance from the nutrient .appeared greatest when idie nitrate reductase levels?were decreasing after reachirtg their maximum value and net protein synthesis 100 had stopped. Therefore, was there sufficient nitrate up take to account for the protein synthesized? Though there was a change of 3-6 ml in the volume of the nutrient, we assumed it remained constant at 50 ml throughout the 14 days in culture. Thus our estimates of nitrate uptake calculated from the observed changes in concentration of nitrate in the nutrient media were slightly low. During the first 5 days the nitrate concentration remained constant or nearly so. The decrease in nitrate concentration that did occur could have provided nitrate nitrogen equivalent to 4.4 to 17,5 mg protein. The instance for which this decrease in nitrate 't 0 T f ,« * ■ • nitrogen was equivalent to 17.5 mg of protein was probably the result of an analytical error, and the 4.4 mg figure was the more typical value. Nitrate must have made a relatively small contribution of nitrogen to protein synthesis during the first five days. In all instances nitrate uptake was evident before soluble protein had reached its maximum val ue. Therefore we took the decrease in nitrate in the cul ture nutrient over days 5-12 and converted it to equivalent protein and compared it to the protein synthesized between days 5-12. We found that the cultures grown on the standard nutrient with 2,4 D showed nitrate disappearance equivalent to 39.4 mg protein and those without 2,4 D showed nitrate disappearance equivalent to 48.1 mg protein. The high phos phate cultures with 2,4 D showed a disappearance of nitrate equivalent to 44.2 mg of protein, while those without 2,4 D 101 showed the equivalent of 83.1 mg protein. Thus over this period sufficient nitrate disappeared from the nutrient to account for the net soluble protein produced (Figure 4 and 11) from days 5-12, except in the case of the high phosphate cultures with 2,4 D. This group was about 3 mg short. There appeared to be sufficient nitrate reductase present in most cases to reduce the nitrate taken up, except for the cultures grown on standard nutrient without 2,4 D. The 3 mg deficit could easily have been made up by the ammonium present, and is less than the error expected in the calculations. At this point we should mention another interesting observation on this sytem. The normal expectation is for nitrate reductase to decline with a drop in nitrate con centration. But we would not have expected it to drop to a very low level while there was still 1 0 mM or greater con centration of nitrate present. Filner (46) used only 2.5 mM nitrate.' In our case some other factor than the pres ence or absence of nitrate was also regulating the nitrate reductase level of the cultures. Currently we have no evi dence concerning what the regulatory mechanism was. Reconsidering the initial hypothesis, we found that, based on the assayed nitrate reductase, growth with 2,4 D did not reduce the level of nitrate reductase and in both cases with and without 2,-4 D ample nitrate reductase was present to account for the soluble protein. However, 102 when comparing nitrate uptake and protein., content .of the cultures' a weak point did develop in that cultures grown in the presence of 2,4 D p a .the.high., phosphate nutrient did not take.up-sufficient nitrate for days 5-12 to account for all of the soluble protein synthesized during that time. The value for nitrate uptake by the cultures in standard nu trient with 2,4 D was low, but adequate to account for the soluble protein, Xf we assume that the nitrate uptake would likewise have been low in the absence of ammonium but in. the presence of 2,4 D, we would have a basis for the ammonium requirement. That is, the ammonium must be pres ent to provide an adequate nitrogen supply which, then, would have ensured an adequate rate of cell division for the production of the embryogenic masses. However, in the absence of data on total nitrogen in the tissue, the model just proposed would not seem an adequate explanation for the large differences in growth rate between the cul tures with and without ammonium. There is the alternative that the nitrate reductase was not functional in vivo. This might have been expected if the phosphate deficiency lowered the.amount, of.DPNH produced.. . However, in the case of the high phosphate cultures the production of DPNH would be expected to be adequate for the in vivo functioning of nitrate reductase, yet markedly greater growth was obtained in the high phosphate nutrient when ammonium was supplied in addition to nitrate. In reality, probably the only way 103 to settle the extent of the dependency on ammonium for an adequate nitrogen supply would be to do a detailed nitro- 15 gen utilization study using N labeling. Faced with the impracticality of such experiments at this time, the effects of tungstate on the growth of the cultures are currently be ing studied to determine the extent to which functional nitrate reductase is of importance. Since no efforts were made to isolate the culture frac tions most likely to give rise to embryos, we can make no comment concerning the differentiation of the tissue in the presence of 2,4 D in relation to their ability to utilize nitrate nor can we be sure that what we have observed reflects changes related to embryogenesis masked in part by the ac tivities of nonembryogenic tissues. Before concluding this section one more possible mech-- anism for the effect of ammonium will be mentioned. At best the explanations we have offered for the ammonium effect do not reflect any great change in the system. How ever, Kanazawa et al. (81), have found a large effect of ammonium on the dark respiration of chlorella. The ammonium ions' primary effect was to activate pyruvate kinase which increased the flow of carbon through the citric acid cycle, increased oxidative phosphorylation, and the synthesis of glutamate. If we assumed that the metabolic activity of the cultures when grown in the absence of ammonium is low and is further blocked by the action of 2,4 D, then 104 activation of pyruvate kinase by ammonium might prove to be one explanation for the growth promoting effects of ammonium. It should be emphasized again that phosphate is prob ably limiting for the wild carrot tissue cultures grown in the original standard nutrient media. Before further work at a biochemical level is carried out on this system the adequacy of the nutrient medium should be fully investigated, and necessary modifications made to sustain growth for longer periods of time. Indeed it appears to be indicated that most tissue culture systems currently in use require re-checking from this point of view. SUMMARY Wetherell and Halperin (2) demonstrated adventive em- bryogenesis in tissue cultures of wild carrot (Daucus carota L.) when nitrogen was supplied'either as ammonium or nitrate. If, however, 2,4 D was present, no embryogenesis could be demonstrated until two conditions were met. First, ammoni- V - urn was obligatory in the initial stages of growth and sec- ond,the growing tissue subsequently had to be transferred to a 2,4 D free medium. These reports of Wetherell and Halperin suggested to us that 2,4 D was inhibiting the forma tion of nitrate reductase necessary for the conversion of nitrate to ammonium in the cultures. If indeed such were the case, the mechanism of action of 2,4 D as an inhibitor of the biosynthesis of nitrate reductase, as suggested by Beevers et al. (3), would be strengthened and also the in volvement of nitrate reductase in embryogenesis suggested . by the work of Wetherell and Halperin could be supported. Accordingly the primary objective of this research was to determine if 2,4 D,does in fact,inhibit the biosynthesis of nitrate reductase in wild carrot tissue cultures. This goal was achieved, but our results showed that 2,4 D did not interfere with the formation of nitrate reductase’. 105 106. We have conducted time course experiments to determine . if the initial requirement for ammonium was the result of a depression of the nitrate reductase level by 2,4 D. In carrying out these experiments it was discovered that the standard nutrient was deficient in phosphate over part of the growth period. For purposes of comparison,‘ a second nutrient containing ten times the phosphate of the standard nutrient was used. The phosphate was not depleted from the nutrient by the termination of the culture period. The use of the higher phosphate nutrient increased fresh weight, protein content, and nitrate reductase levels of the cultures. Cultures grown without 2,4 D had higher fresh weights than cultures grown on the same nutrient medium with 2,4 D. Embryogenesis was observed in all cultures meeting the two conditions mentioned above. In the case of the cul tures grown on the higher phosphate nutrient medium, more phosphate and nitrate disappeared after day 9 from the nutri ent medium of the cultures without 2,4 D than from those with 2,4 D. Our preliminary experiments indicated that the reported requirement for ammonium during the growth of ■ cultures with 2,4 D to achieve embryogenesis in the absence of 2,4 D may not be absolute. The nitrate reductase levels changed with time as did the specific activity of the nitrate reductase. The nitrate reductase levels were adequate to account for the soluble protein synthesized by the cultures from day 5 on. After 107 the first 5 days adequate nitrate was taken up by the cul tures to provide nitrogen for the synthesis of soluble pro tein. except in one case. This exception was for cultures grown on the higher phosphate medium with 2,4 D. Supply ing adequate nitrogen for protein synthesis for the first 5 days in culture in the presence of 2,4 D may have been one of the essential functions of ammonium in the standard nutrient media. However, this explanation for the ammoni um requirement seems insufficient to explain the difference in growth between cultures grown on nitrate alone compared to those supplied with both ammonium and nitrate as more than enough nitrate was present to supply the nitrogen needed. It appears that the next major advance in this system should be the development of a more optimal nutrient medium so that biochemical events related to embryogenesis may be studied without unexpected and complicating effects of unanticipated nutrient deficiencies. In addition, there remains the question of what controls the nitrate reductase level besides nitrate in the nutrient. 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