- Email: [email protected]
Release of enzymes by plant tissue cultures
Lt~e Sciences Ho . 1, pp " 50-62 , 1963 . Parrg~on Presa~ Ina.. Printed in the IInited States .
RELEAâE OF ENZYt~S BY PLANT TISSUE CULTURFS~ Jsaob Straus~ and W.A~ Campbells Deparl~snt of Biology, University of Oregon, Eugene, Oregon (Received 7 December 1962)
Reports of the release of enzymes into nutrient media by tissue cultures 2, 3,4, 5, 6 . of higher plants have appeareii in the literaturel' Many of these reports are concerned with tissue cultures derived from diseased plants . Thus Lipetz and Galaton 3 and Witham and Gentile 6 used tissue cultures derived from crown-gall tumors of Parthenociasus . Nickell and Brakke 4 utilized tissue cultures derived from Rumex virus tumors ; and one of the tissues reported upon by Reinert, et al . 5 was derived from Picea q l auca tumors of unknown etiology . In an attempt to discover whether the release of enzymes by plant tissue cultures was of common occurrence not only among tissues derived from diseased plants, but from presumably healthy ones as well, we undertook a survey of ten tissue cultures, only one of which is known to have been derived from a diseased plant . This particular tissue is derived from Nicotiana tabacum and is designated as M-222 . It was isolated by G . Morel from TMV-infected plants, but is no longer thought to be infected with the virus . This report supplies data which indicate that the release of enzymes by tissue cultures of higher plants is probably of common occurrence . Materiale and Methods All of the tissues utilized in this study except for the maize endosperm tissues were routinely grown on a modified White's agar 7 supplemented with 20 °Jo
(vw)
coconut water and 2 mg~l 2, 4-dichlorophenoxyacetic acid. The endos-
perm tissues were grown on modified White's agar containing 2 g~l 1-asparagine . The tissue cultures of Roea, Pelartlonium , Lycopersicon, and the Nicotianas ~ Work supported in part by PE3 grant RG-7291 . ~ Temporary address until June 1963 : Laboratoire do Phytotron, CNRS, GiP-surYvstts (S . et 0 . ), France . $ National Sd.snae Foundation Undergraduate Rssesroh Scholar. 50
OF B118II~3 BY PLAiR TISSQE COLTÜRBS
No° 1
51
were derived from the stems of the respective plants . The tissue culture of Cupressus was derived from the axis of the staminate cone ; while those of Zea were derived from the endosperm . For further details on the sources of these tissues, see reference 8 . All the tissues, except for the endosperme were tented after 30-35 days growth . The endosperms were tested after 20-25 days growth . Assays Peroxidase.
It was not possible to use pyrogallol as the chromogenic
agent as we obtained a variety of colors ranging from blue, purple, green and orange when testing the crude enzyme preparations . Instead we used o-dianisidine . One ml of a 1 °J° solution of o-dianisidine in absolute m~anol was added to 99 ml water . Reaction mixtures contained the following : 1 ml of the aqueous chromogenic agent, Z ml phosphate buffer (0 . 05 M final concentration at PH 6 . 1) and 1 ml of the enzyme preparation . The enzyme preparation was diluted 1 : 50 with water before use . This was necessary because of the intensity of the color which developed . At time zero, 1 ml 40 mM hydrogen peroxide was added and the contents of the tube were mixed by lateral shaking . At the end of 7 . 5 minutes, 5 ml of 5 % trichloroacetic acid were added to stop the reaction . The color density was determined with a Klett colorimeter equipped with a blue filter (# 42) . No attempt was made to convert the Klett units to P. Z . units . Thus, we realize that the data presented here can only be used for comparative purposes among the particular tissues studied and cannot be applied quantitatively to data in other literature. Acid phoaphatase .
The chromogenic agent was prepared by dissolving
0 .42 g disodium p-nitrophenyl phosphate (Sigma brand) in 100 ml water . Reaction mixtures contained 1 ml chromogenic agent solution, 1 ml acetate buffer (0 .05 M final concentration at pH 5 . 0) ; and 2 ml enzyme preparation which had previously been diluted 1 : 10 with water . The mixture was incubated in a water bath shaker maintained at 30 ° C. At the end of 30 minutes, 6 ml of 0 .25
/o
NaOH were added
to develop the color . The color density was determined with a Klett colorimeter utilizing a blue filter (#42) . Klett unite were converted into absolute units (~tmoles of p-nitrophenol) by comparison with a calibration curve prepared with known amounts of p-nitrophenol . This procedure is a modification of Lowry's method 9 .
~2
ßBIFJ83 Olr E1TZ~8 BY PI~ ?I38~ COL?ü1~8 Iadoleacetic acid (IAA) o~xidase .
lfo. 1
Reaction mixtures were composed of the
following : 1 ml each of IAA, MnC12 , and dichlorophenol (final concentration in the reaction mixture of all three was 10-4M), 6 ml phosphate buffer (0 . 05 M final concentration at pH 6.1) and 1 ml of the undiluted enzyme preparation . Immedia-
tely upon addition of the enzyme aad thorough mixing, 1 ml aliquots were removed aad tested immediately with Gordon aad Weber's modified Salkowski reagent 10 to determine whether substances iaterferriag with the Salkowaki reaction were present. The nemainder of the reaction mixtures was incubated in a water bath shaker maintained st 30' C . At the end of one hour, 1 ml aliquots of the incubation mixtures were added to 5 ml of the modified Salkowski reagent . The color was
allowed to develop for 25 minutes . The color density was determined with a Klett colorimeter equipped with a green filter (# 54) . Klett units were converted into /moles of IAA by comparison with a calibration curve previously prepared with known amounts of IAA. Amvlase . Aaaylase activity was determined by incubating the enzyme preparations with starchand subsequent testing for as increase is reducing power using Beaedict's quantitative method 11 . Reaction mixtures contained 1 ml 1
°Je
aqueous soluble starch solution, 3 ml phosphate buffer (0 .05 M final conc, at PH 6 .1) aad 1 ml undiluted enzyme preparation . The mixture was incubated for 2 hours on a water bath shaker at 30'C . One ml aliquots were subsequently analyzed for increases in reducing power . The blank values for most of the
enzyme preparations were quite high and coasqueatly we do not place too much reliance oa the absolute figures obtained . However, these data are included in this report because there was a consistency is the behaviour of the tissues when
soaked in calcium solutions (see the section oa results) . Klett units were converted into ~aoles of glucose by comparing them to a calibration curve prepared
with lmown amounts of glucose . Hence, the increase in reducing power ie expressed as pnnoles ôf glucose .
Results The Medium The media upba which samples of tissue lied been grown were tested
qualitatively for the four enzymes . The agar contents (after careful removal
Xo. 1
ßEIF~ Oh ~ZII~S BZ PL1R TISSt~ COLT~S
53
of all the tissue) of five vials of medium (approx . 50 ml) were removed and homogenised with an aqual volume of water is a YirTie homogeaizer .
The
mixture was filtered through paper with suction and the filtrate utilised as the enzyme preparation, positive results were obtained for all ten tissues for perozidase, amylase, and acid phosphatase . No IAA oxidaee, however, could be detected in the medium for any of the tissues . Unwashed Tissue Five grams of tissue were placed directly into 25 ml deioaized water and pbrmitted to steep with occasional shaking at room temperature . At the end of one hour, the mixture was filtered and the filtrate assayed for the four enzymes . The data are summarized in table 1. It ie apparent that all the tissues released appreciable amounts of phosphatase, peroxidaee and amylase . Two of the tobacco tissues alèo exhibited relatively high IAA oxidaee activity also. It is interesting to note that the three endosperm tissues are well distinguished from all the others by their very high phosphatase activity . Wsshed Tissue Since enzyme activity was found in the medium, it seemed probable that some, if perhaps not all, of the enzyme activity recovered from the unwashed tissue when soaked in water was simply due to an accumulation of enzyme os the tissue surfaces during the several-week growth period and did not truly represent the amounts of enzyme released by the tissue during the one-hour soaking period . Therefore, enzyme preparations were prepared with previously washed tissue . Five grams of tissue were placed in a cheesecloth bag and swirled in 600 ml water. This was repeated five more times with fresh 600 ml volumes of water . The washed tissue vRas then steeped as usual in 25 ml water for one hour . The mixture was filtered ahd the filtrate assayed for enzyme activity . The data are contained in table 2 . Activities of phoephataee, p~eroxidaee, and amylase were drastically reduced in every instance except for a slight increase in peroxidaee activity for Cupreseus . However, interestingly enough, IAA oxidase activity was increased for several of the tissues :. It appears then, at least for the first three mentioned ,easymes that there is a considerable accumulation oa the surfaces of the tissues during the growth period, It ie a little difficult to explain the increased IAA oxidase activity exhibited by several of the washed tissues . Ose possibility is that
~T~ OF ENZII~3 BY PIJIIfr TI3S~ CIILTÜRES
54
No. 1
Table 1 Activity of Enzymes Obtaiaed Prom Unwashed Tissue Soaked in Water
Tissuea
Phosphataseb
Peroxidase c
Amylase d
IA.A Oxidasee
1
208
48
29
0
2
715
29
304
30
3
376
159
240
0
4
493
109
165
31
5
750
200
103
175
6
580
181
196
119
7
5660
158
36
0
8
7900
167
31
0
9
7800
155
31
0
10
1420
80
92
46
a . Tissues : 1 , Roea multiflora Thunb . ; 2, Pelarttonium sp . ; 3, Lycopersicon esculentum Mill . ; 4, Nicotiana tabacum L . (M-222) ; 5, N, tabacum L. (H-239) ; 6, N. tabacum L . (H-196) ; 7, Zea maos L . (Pl) ; 8, Z . mctus L. (Pl-C) ; 9, Z, maus L . (Pl-R) ; 10, Cupressus funebris Endl . b . Phosphatase : micromoles (x 10-3) of p-nitrophenol released from p-nitrophenyl phosphate in 30 minutes . c . Peroxidase : Klett units at the end of 7 . 5 minutes . d . Amylase : increase in reducing power in 2 hours expressed as micromoles of glucose . e . IAA oxidase : micrograms of IAA destroyed in one hour . Reaction mixtures contain 175 micrograms IAA initially .
Iiar~~ OF B11Z~3 BI PhA1~ TI33IIE CIIISÜI~
llo, 1
55
Table 2 Activity of Enzymes Obtained From Washed Tissue Soaked in Water
Tissuea
Phosphataeeb
Peroxidasec
Amylased
Oxidaae e
TA A
1
8
46
3
0
2
76
72
111
175
3
20
74
2
135
4
14
31
2
122
5
22
47
0
175
6
4
31
2
110
7
218
32
41
0
8
274
42
20
9
17b
30
15
59
10
---
113
8
0 0
For explanation of the superscripts, refer to table 1,
an inhibitor or denaturant of the enzyme is also released, but at a slower rate than the enzyme. Thus, this substance would accumulate during the relatively long growth period, but not during the one-hour soaking period . For a loag time it has been impossible to demonstrate TA A oxidase activity in tissue cultures . In 1957, however, Reinert, et al . 5 reported the release of IAA oxidase by tissue cultures derived from pea roots and tumors of Picea . More recently, Witham and Gentileb have demonstrated the same thing to occur with tissue cultures derived from crown-gall tumors of Parthenociasue . Straus and Gerdingl 2 have reported on the release of as extremely active IAA oxidase from tissue cultures derived from normal E
edra plants . The data in the present
report add several more tissues to the list of tissue cultures which release active IAA oxidase .
56
RBIFASg~ OF EèiZYlO;S BY PLAIPP TI33UE ~IIRB3
Bo. 1
Table 3 Activity of Enzymes Obtained From Washed Tissue Soaked in 0 . 05 M Calcium Chloride
Tissues
Phoephataseb
Peroxidasec . Amylase d
IAA Oxidasee
1
0
117
0
20
2
22
114
---
113
3
8
145
0
175
4
24
92
0
122
5
14
141
0
175
6
0
121
0
175
7
384
187
25
175
8
300
127
9
175
9
458
174
7
175
10
80
174
0
124
For explanation of the superscripts, refer to table 1 . Washed Tissue Soaked in 0 . 05 M Calcium Chloride We considered the fact that deionized water is not a medium which might be expected to maintain the integrity of the plasma membranes of the tissue cells . Calcium ions are considered to be essential to prevent leakinees of such membranes We therefore repeated the "leakage" experiments by soaking the tissue in a solution of 0 . 05 M calcium chloride. The tissue was first washed in the usual manner and then soaked in the calcium solution for one hour . The tissue was filtered off and the filtrate tested for enzyme activity. The results are summarized in table 3 . Contrary to what we expected, increased activities of certain of the enzymes were recovered from such filtrates . Peroxidase activity was considerably enhanced only in the preparations of Pl and Pl-R and to a lesser degree for Pl-C and M-22 . Amylase activity, however, was diminished in all cases . The moat remarkable finding was that those filtrates from tissues which had not previously exhibited any
aBIaJ~ 0~ SUZII~S BI PIJäT ?I330B C0'LTUR88
llo. 1
S7
Table 4 IAA Ozidase Activity in Homogenates of Tissues Compared to Calcium Solution "Leakage"
Tissues
Homogenaatee
Calcium Filtrates °
Untreated Dialyzed
From Table 3
1
0
0
20
2
137
113
3
0
91
0
175
4
0
0
122
5
0
0
175
6
0
28
175
7
0
0
175
8
0
0
175
9
0
0
175
10
0
0
124
For explanation of the superscripts, refer to table 1 . TAA
oxidsse activity is filtrates from water-soaked tissues, now exhibited rather
high IAA oxidase activities . The only exceptioa was the Rosa tissue which showed only a small amouat of activity . IAA Oxidase Activity in Homogenates When we fouad that all of the tissues "leaked" IAA oxidase into the calcium
chloride solution, we werm quite surprised as it has been very difficult to detect
IAA oxidase in tissue cultures as mentioned above . Indeed, there are only negative
reports of such activity in homogenates of tissue cultures (see ref, 3, for example),
Therefore, it seemed to be of interest see whether homgenates of some of the tissues uader iavestigstioa exhibited oxidase activity . Five grams of tissue were homogenized in 20 ml water in a YirTis homogenizer, The brei was centrifuged at
58
RBIBL~IS OF SNZYt~B BY PLAIaT TI83IIS CIILTÛR&3
No. 1
Table 5 IAA Oxidase and Phosphatase Activities Obtained from Soaking Cell Wall Preparations in 0 . 05 M Calcium Chloride
Tissue s
Phosphatase b Cell Wall Homogenates
IAA Oxidase e
2
540
2000
175
3
375
900
175
4
430
1000
175
7
1340
18,000
175
8
1200
9,500
175
9
1540
18,000
175
For explanation of the superscripts, refer to table 1 . 20, 000 x G for 20 minutes . The supernatant was made up to 25 ml aad assayed in the usual manner for IAA oxidase . Aliquots of the same supernatants were also dialyzed in the cold against several changes of distilled water for 24 hours and also aaalyzed . Table 4 summarizes the results . For ease in comparison, the data for TAA oxidase activity contained in table 3 are also included . Only one tissue homo-
genate, that of PelarQonium, exhibited any oxidase activity. The dialyzed homogenate of H-196 contained some small activity. These results confirm those of other workers in that they indicate the extremely rare occurrence of IAA oxidase activity in tissue culture homogenates . Nevertheless, we are still faced with the evidence which indicates that these tissues can release IAA oxidase in the presence of calcium ions and, indeed some of them in the presence only of water . Thus, either the enzyme (one or more components as suggested by Lipetz and Galston 3 ) is destroyed by the homogenizing procedure or, an undialyzable inhibitor or a denaturant is released or produced by
lfo . 1
ItE?E~S~ OF ~IZâ~3 HY ?LA1R ?~SSIIE CIILTtIit$S
59
the process . However, the much milder soaking procedure does not inactivate the enzyme nor release or activate inhibitors . Why tissue cultures should be so unusual with respect to homogenates is as interesting problem since homogenates of organs of intact plants frequently have high I1~A oxidase activities .
Activities Rec overed From Cell Wall Preparations . Ia order to ascertain whether the enzymes were in fact bound to the cell walls and then released bY the calcium ions, cell wall preparations of some of the tissues were tested. Five grams of tissue in 25 ml water were homogenized in a VirTis homogenizer for a full minute at top speed . This procedure ensures about 90 % disrupted cells $ . The brei was centrifuged at low speed (470 x G) a.ad the supernatant discarded . Twenty ml water were added to the residue in the tube and the contents vigorously stirred and again centrifuged at low speed. This was repeated 3 more times so that the cell wall preparations were washed 4 times . The washed residue was then suspended in 25 ml 0 .05 M CaC12 and incubated at room temperature on a reciprocal shaker, At the end of one hour, the mixture was centrifuged and the supernatant assayed for enzyme activity. Only IAA oxidase and acid phosphatase activities were determined for nix of the tissues . The cell wall preparation supernatants of the six tissues all showed maximum IAA oxidase activity (table 5). Since the reaction mixtures ~~n s'n d 1 751wg IAA sad all of it waé destroyed, it is impossible to determine the relative activities of the preparations . What can be said is that all six tissues exhibited high IAA oxidaee activities . The acid phoephatase activity exhibited by these preparations is tremendously greater than that recovered from calcium filtrates in which intact tissue had been soaked. For comparison, data obtained from phosphataee assays of homogenates of these tissues are also included in table 5 . The homogenates of these six tissues contain high phosphatase activities . Thus, it would appear that relatively little of the enzyme leaks out except perhaps in the case of the three endosperm tissues (table 2). Nevertheless, the supernatants from the calcium-soaked cell
wall
prepa-
rations exhibit very high activities . It seems reasonable to assume that the cell walls bind the enzyme during the homogenizing procedure and release it in the presence of calcium ions, The same thing is probably true of IAA oxidase . The simplest interpretation of the enhanced release bY the intact tissues of certain . of the enzymes -by calcin:a ions is that gees enzymes are continously being
60
R81a1S6 ~Oh BüZII~.9 BY PL~11T TISSQB CQLTUi~3
No. 1
released by the tissues in greater or lesser amounts . Some of the enzyme is bound to the wall or, possibly the outer membrane surface and the calcium ions serve to release the enzyme from the binding sites . Japsen, et al . 13 have shown that cell wall preparations of Ay~aa coleoptilee do in fact bind enzymes . With regard to amylase and phosphatase (except for the three eadosperms and M-222), it is probable that these enzymes are not released is very great amounts by the tissue cells . Thus, if the calcium ions do indeed serve to preserve the integrity of the plasma membranes, then the enzyme activities recovered from the filtrates of the washed tissue soaked in CaC12 solution represent at least a portion of the quantity of enzyme which had previously been released by the tissue and had been bound to the cell wall . Discussion An important question, of course, is the mechanism of enzyme release by these tissues . Are they released by dead or dying sells ; are they actively secreted ; or do they diffuse out passively ? The data presented in this report do not permit any unequivocal conclusions to be drawn in this regard. Other workers 1 , 2,14~ve presented evidence which indicates that the release of amylase by tissue cultures of higher pleats is not dependent upon moribund cells . However, except for the intrinsic interest of the problem of enzyme release, the important fact is that enzymes are is some manner released from the cytoplasm into the medium upon which the tissues are grows or are bound to the outer surfaces of the cells . A tissue culture of a higher pleat has a different set of "problems to solve", teleologically speaking, than the intact plant from which it has been derived . Given minerals, atmospheric gases, and water, the intact green plant synthesizes all of its metabolites . Additionally, the vascular plants have spedalized transport systems fpr the movement of the elaborated metabolites to a]1 parts of the plant . Tissue cultures, on the other head, must be supplied not only with minerals, atmospheric gases, and water, but frequently with auxin, vitamins, and an organic nitrogen source . These substances must the taken up by the cells in one form or another and must also be transported to cells which are not directly in contact with the nutrient medium . The situation is analogous to that of a relatively massive fungal mycelium.
xo . ~
asia~sa og xxzn~s
Bi Pt exr ~iss~ cor~tfxBS
61
Uader certain circumstances, plant tissue cultures must be able to modify the substratum in order to grow . For e~tsmple, it is known that a number of tissue cultures are able to grow on media in which the sole carbon source supplied is 1,2,4,? . starch Investigation has shown that such tissue cultures do release an 1,2,4 . amylase into the medium Thus, the ability to digest starch externally is
essential for these tissues when placed on a starch medium. Recently, Straus8 has
shown that the inversion of sucrose in nutrient media by eleven different tissue cultureè is carried out by invertase bound to the cell walls of the tissues . Straus and Gerdingl2 have reported evidence which stroagly indicates that the inability of
auxin-requiring tissue cultures derived from Ephedra to grow on media in which the exogenous auxin is IAA is due to the release of IA .A oxidase by the tissues . The tissues grow very well on media containing synthetic "unnatural" auxins . Hence, it does aot seem to be inconceivable that the release of various
enzymes (by whatever means) by the tissue cultures into the medium modifies the medium in some manner and, in some cases is essential to the growth of the tissues (e, g. , when placed oa starch media) and in others prevents the growth of the tissues (as in the case of E
edra . It is tempting to speculate, although
there is no evidence on the point, that the explanation for the difficulty or impossibility of deriving tissue cultures from moat monocot species and some dicot
and gymaosperm species lies in the inability of the original expla.nt to suitably modify the artificial nutrient medium or, conversely the medium may be detrimentally modified . Summary Data are presented which indicate that tissue cultures of higher planta release enzymes into the medium upon which they are grown . Some of the enzyme released by the tissues appears to be bound to the cell wall. Release
of indoleacetic acid o~cidase (IAA oxidase) and peroxidase ie enhanced when the tissues are soaked in 0 . 05 M CaC12 . Cell wall preparations of the tissues when incubated with 0 . 05 M CaC12 release more TA A oxidase and acid phosphatase than do the intact tissues . Thus, it would appear that the cell walls bind the enzymes during the homogenizing procedure and the calcium ions probably serve to release the enzymes from the binding sites. One of the most interesting facts uncovered during this study is the demonstration of
G2
REIFA.gS OF ENZ~3 BZ PLA1rP ?I3SÜS CULTIIR$3
llo . 1
the release of IA.A oxidase by all 10 of the tissues investigated when the tissues are soaked in 0 .05 !VI CaC1 2 . It is tentatively suggested that the
release of enzymes by the tiasuee~ may serve to modify the nutrient medium to affect the growth of the tissues . References 1.
F . CONSTABEL, Pla.ata . 57, 331 (1961) .
2.
W. H . K. KARSTENS and VERA DE MEESTER-MANGER CATS, Acta Bot. Neerl . 4, 263 (1960)
3.
J. LIPETZ and A . W. GAISTON, Amer . Jour . Bot . ~j 193 (1959)
4 . L. G . NICKELL and M . K. BRAKKE, Amer . Jour . Bot . 4~1, 390 (1954) 5.
J. REINERT, H . SCHRAUDOLF and M . TAZAWA, Naturwiss . 4~, 588 (1957) .
6.
F . H . WITHAM and A. C . GENTILE, Jour. Exp. Bot . lj 188 (1960)
7.
J . STRAUS and C. D. LARDE, Amer . Jour . Bot. 41, 687 (1954) .
8.
J . STRAUS, Plant Physiol . _, 342 (1962)
9.
O. H . LOWRY, in Methods in Eazymolorxy, ~ Academic Preas, New York (1957) .
10 . S. A . GORDON and R. P. WEBER, Plant Physiol . 26, 192 (1951) . 11 . S. R . BENEDICT, Jour . Biol . Chem . 76, 457 (1928) . 12 . J . STRAUS and R . K. GERDING, Plant Physiol. 3? suppl . , xiv (1962) . 13. E . F . JANSEN, ROSIE JANG, and J. BONNER, Plant Physiol. 567 ( 1960) . 14 . M. K. BRAKKE and L. G. NICKELL, Ann . Biol. 31, 215 (1955) .
RELEAâE OF ENZYt~S BY PLANT TISSUE CULTURFS~ Jsaob Straus~ and W.A~ Campbells Deparl~snt of Biology, University of Oregon, Eugene, Oregon (Received 7 December 1962)
Reports of the release of enzymes into nutrient media by tissue cultures 2, 3,4, 5, 6 . of higher plants have appeareii in the literaturel' Many of these reports are concerned with tissue cultures derived from diseased plants . Thus Lipetz and Galaton 3 and Witham and Gentile 6 used tissue cultures derived from crown-gall tumors of Parthenociasus . Nickell and Brakke 4 utilized tissue cultures derived from Rumex virus tumors ; and one of the tissues reported upon by Reinert, et al . 5 was derived from Picea q l auca tumors of unknown etiology . In an attempt to discover whether the release of enzymes by plant tissue cultures was of common occurrence not only among tissues derived from diseased plants, but from presumably healthy ones as well, we undertook a survey of ten tissue cultures, only one of which is known to have been derived from a diseased plant . This particular tissue is derived from Nicotiana tabacum and is designated as M-222 . It was isolated by G . Morel from TMV-infected plants, but is no longer thought to be infected with the virus . This report supplies data which indicate that the release of enzymes by tissue cultures of higher plants is probably of common occurrence . Materiale and Methods All of the tissues utilized in this study except for the maize endosperm tissues were routinely grown on a modified White's agar 7 supplemented with 20 °Jo
(vw)
coconut water and 2 mg~l 2, 4-dichlorophenoxyacetic acid. The endos-
perm tissues were grown on modified White's agar containing 2 g~l 1-asparagine . The tissue cultures of Roea, Pelartlonium , Lycopersicon, and the Nicotianas ~ Work supported in part by PE3 grant RG-7291 . ~ Temporary address until June 1963 : Laboratoire do Phytotron, CNRS, GiP-surYvstts (S . et 0 . ), France . $ National Sd.snae Foundation Undergraduate Rssesroh Scholar. 50
OF B118II~3 BY PLAiR TISSQE COLTÜRBS
No° 1
51
were derived from the stems of the respective plants . The tissue culture of Cupressus was derived from the axis of the staminate cone ; while those of Zea were derived from the endosperm . For further details on the sources of these tissues, see reference 8 . All the tissues, except for the endosperme were tented after 30-35 days growth . The endosperms were tested after 20-25 days growth . Assays Peroxidase.
It was not possible to use pyrogallol as the chromogenic
agent as we obtained a variety of colors ranging from blue, purple, green and orange when testing the crude enzyme preparations . Instead we used o-dianisidine . One ml of a 1 °J° solution of o-dianisidine in absolute m~anol was added to 99 ml water . Reaction mixtures contained the following : 1 ml of the aqueous chromogenic agent, Z ml phosphate buffer (0 . 05 M final concentration at PH 6 . 1) and 1 ml of the enzyme preparation . The enzyme preparation was diluted 1 : 50 with water before use . This was necessary because of the intensity of the color which developed . At time zero, 1 ml 40 mM hydrogen peroxide was added and the contents of the tube were mixed by lateral shaking . At the end of 7 . 5 minutes, 5 ml of 5 % trichloroacetic acid were added to stop the reaction . The color density was determined with a Klett colorimeter equipped with a blue filter (# 42) . No attempt was made to convert the Klett units to P. Z . units . Thus, we realize that the data presented here can only be used for comparative purposes among the particular tissues studied and cannot be applied quantitatively to data in other literature. Acid phoaphatase .
The chromogenic agent was prepared by dissolving
0 .42 g disodium p-nitrophenyl phosphate (Sigma brand) in 100 ml water . Reaction mixtures contained 1 ml chromogenic agent solution, 1 ml acetate buffer (0 .05 M final concentration at pH 5 . 0) ; and 2 ml enzyme preparation which had previously been diluted 1 : 10 with water . The mixture was incubated in a water bath shaker maintained at 30 ° C. At the end of 30 minutes, 6 ml of 0 .25
/o
NaOH were added
to develop the color . The color density was determined with a Klett colorimeter utilizing a blue filter (#42) . Klett unite were converted into absolute units (~tmoles of p-nitrophenol) by comparison with a calibration curve prepared with known amounts of p-nitrophenol . This procedure is a modification of Lowry's method 9 .
~2
ßBIFJ83 Olr E1TZ~8 BY PI~ ?I38~ COL?ü1~8 Iadoleacetic acid (IAA) o~xidase .
lfo. 1
Reaction mixtures were composed of the
following : 1 ml each of IAA, MnC12 , and dichlorophenol (final concentration in the reaction mixture of all three was 10-4M), 6 ml phosphate buffer (0 . 05 M final concentration at pH 6.1) and 1 ml of the undiluted enzyme preparation . Immedia-
tely upon addition of the enzyme aad thorough mixing, 1 ml aliquots were removed aad tested immediately with Gordon aad Weber's modified Salkowski reagent 10 to determine whether substances iaterferriag with the Salkowaki reaction were present. The nemainder of the reaction mixtures was incubated in a water bath shaker maintained st 30' C . At the end of one hour, 1 ml aliquots of the incubation mixtures were added to 5 ml of the modified Salkowski reagent . The color was
allowed to develop for 25 minutes . The color density was determined with a Klett colorimeter equipped with a green filter (# 54) . Klett units were converted into /moles of IAA by comparison with a calibration curve previously prepared with known amounts of IAA. Amvlase . Aaaylase activity was determined by incubating the enzyme preparations with starchand subsequent testing for as increase is reducing power using Beaedict's quantitative method 11 . Reaction mixtures contained 1 ml 1
°Je
aqueous soluble starch solution, 3 ml phosphate buffer (0 .05 M final conc, at PH 6 .1) aad 1 ml undiluted enzyme preparation . The mixture was incubated for 2 hours on a water bath shaker at 30'C . One ml aliquots were subsequently analyzed for increases in reducing power . The blank values for most of the
enzyme preparations were quite high and coasqueatly we do not place too much reliance oa the absolute figures obtained . However, these data are included in this report because there was a consistency is the behaviour of the tissues when
soaked in calcium solutions (see the section oa results) . Klett units were converted into ~aoles of glucose by comparing them to a calibration curve prepared
with lmown amounts of glucose . Hence, the increase in reducing power ie expressed as pnnoles ôf glucose .
Results The Medium The media upba which samples of tissue lied been grown were tested
qualitatively for the four enzymes . The agar contents (after careful removal
Xo. 1
ßEIF~ Oh ~ZII~S BZ PL1R TISSt~ COLT~S
53
of all the tissue) of five vials of medium (approx . 50 ml) were removed and homogenised with an aqual volume of water is a YirTie homogeaizer .
The
mixture was filtered through paper with suction and the filtrate utilised as the enzyme preparation, positive results were obtained for all ten tissues for perozidase, amylase, and acid phosphatase . No IAA oxidaee, however, could be detected in the medium for any of the tissues . Unwashed Tissue Five grams of tissue were placed directly into 25 ml deioaized water and pbrmitted to steep with occasional shaking at room temperature . At the end of one hour, the mixture was filtered and the filtrate assayed for the four enzymes . The data are summarized in table 1. It ie apparent that all the tissues released appreciable amounts of phosphatase, peroxidaee and amylase . Two of the tobacco tissues alèo exhibited relatively high IAA oxidaee activity also. It is interesting to note that the three endosperm tissues are well distinguished from all the others by their very high phosphatase activity . Wsshed Tissue Since enzyme activity was found in the medium, it seemed probable that some, if perhaps not all, of the enzyme activity recovered from the unwashed tissue when soaked in water was simply due to an accumulation of enzyme os the tissue surfaces during the several-week growth period and did not truly represent the amounts of enzyme released by the tissue during the one-hour soaking period . Therefore, enzyme preparations were prepared with previously washed tissue . Five grams of tissue were placed in a cheesecloth bag and swirled in 600 ml water. This was repeated five more times with fresh 600 ml volumes of water . The washed tissue vRas then steeped as usual in 25 ml water for one hour . The mixture was filtered ahd the filtrate assayed for enzyme activity . The data are contained in table 2 . Activities of phoephataee, p~eroxidaee, and amylase were drastically reduced in every instance except for a slight increase in peroxidaee activity for Cupreseus . However, interestingly enough, IAA oxidase activity was increased for several of the tissues :. It appears then, at least for the first three mentioned ,easymes that there is a considerable accumulation oa the surfaces of the tissues during the growth period, It ie a little difficult to explain the increased IAA oxidase activity exhibited by several of the washed tissues . Ose possibility is that
~T~ OF ENZII~3 BY PIJIIfr TI3S~ CIILTÜRES
54
No. 1
Table 1 Activity of Enzymes Obtaiaed Prom Unwashed Tissue Soaked in Water
Tissuea
Phosphataseb
Peroxidase c
Amylase d
IA.A Oxidasee
1
208
48
29
0
2
715
29
304
30
3
376
159
240
0
4
493
109
165
31
5
750
200
103
175
6
580
181
196
119
7
5660
158
36
0
8
7900
167
31
0
9
7800
155
31
0
10
1420
80
92
46
a . Tissues : 1 , Roea multiflora Thunb . ; 2, Pelarttonium sp . ; 3, Lycopersicon esculentum Mill . ; 4, Nicotiana tabacum L . (M-222) ; 5, N, tabacum L. (H-239) ; 6, N. tabacum L . (H-196) ; 7, Zea maos L . (Pl) ; 8, Z . mctus L. (Pl-C) ; 9, Z, maus L . (Pl-R) ; 10, Cupressus funebris Endl . b . Phosphatase : micromoles (x 10-3) of p-nitrophenol released from p-nitrophenyl phosphate in 30 minutes . c . Peroxidase : Klett units at the end of 7 . 5 minutes . d . Amylase : increase in reducing power in 2 hours expressed as micromoles of glucose . e . IAA oxidase : micrograms of IAA destroyed in one hour . Reaction mixtures contain 175 micrograms IAA initially .
Iiar~~ OF B11Z~3 BI PhA1~ TI33IIE CIIISÜI~
llo, 1
55
Table 2 Activity of Enzymes Obtained From Washed Tissue Soaked in Water
Tissuea
Phosphataeeb
Peroxidasec
Amylased
Oxidaae e
TA A
1
8
46
3
0
2
76
72
111
175
3
20
74
2
135
4
14
31
2
122
5
22
47
0
175
6
4
31
2
110
7
218
32
41
0
8
274
42
20
9
17b
30
15
59
10
---
113
8
0 0
For explanation of the superscripts, refer to table 1,
an inhibitor or denaturant of the enzyme is also released, but at a slower rate than the enzyme. Thus, this substance would accumulate during the relatively long growth period, but not during the one-hour soaking period . For a loag time it has been impossible to demonstrate TA A oxidase activity in tissue cultures . In 1957, however, Reinert, et al . 5 reported the release of IAA oxidase by tissue cultures derived from pea roots and tumors of Picea . More recently, Witham and Gentileb have demonstrated the same thing to occur with tissue cultures derived from crown-gall tumors of Parthenociasue . Straus and Gerdingl 2 have reported on the release of as extremely active IAA oxidase from tissue cultures derived from normal E
edra plants . The data in the present
report add several more tissues to the list of tissue cultures which release active IAA oxidase .
56
RBIFASg~ OF EèiZYlO;S BY PLAIPP TI33UE ~IIRB3
Bo. 1
Table 3 Activity of Enzymes Obtained From Washed Tissue Soaked in 0 . 05 M Calcium Chloride
Tissues
Phoephataseb
Peroxidasec . Amylase d
IAA Oxidasee
1
0
117
0
20
2
22
114
---
113
3
8
145
0
175
4
24
92
0
122
5
14
141
0
175
6
0
121
0
175
7
384
187
25
175
8
300
127
9
175
9
458
174
7
175
10
80
174
0
124
For explanation of the superscripts, refer to table 1 . Washed Tissue Soaked in 0 . 05 M Calcium Chloride We considered the fact that deionized water is not a medium which might be expected to maintain the integrity of the plasma membranes of the tissue cells . Calcium ions are considered to be essential to prevent leakinees of such membranes We therefore repeated the "leakage" experiments by soaking the tissue in a solution of 0 . 05 M calcium chloride. The tissue was first washed in the usual manner and then soaked in the calcium solution for one hour . The tissue was filtered off and the filtrate tested for enzyme activity. The results are summarized in table 3 . Contrary to what we expected, increased activities of certain of the enzymes were recovered from such filtrates . Peroxidase activity was considerably enhanced only in the preparations of Pl and Pl-R and to a lesser degree for Pl-C and M-22 . Amylase activity, however, was diminished in all cases . The moat remarkable finding was that those filtrates from tissues which had not previously exhibited any
aBIaJ~ 0~ SUZII~S BI PIJäT ?I330B C0'LTUR88
llo. 1
S7
Table 4 IAA Ozidase Activity in Homogenates of Tissues Compared to Calcium Solution "Leakage"
Tissues
Homogenaatee
Calcium Filtrates °
Untreated Dialyzed
From Table 3
1
0
0
20
2
137
113
3
0
91
0
175
4
0
0
122
5
0
0
175
6
0
28
175
7
0
0
175
8
0
0
175
9
0
0
175
10
0
0
124
For explanation of the superscripts, refer to table 1 . TAA
oxidsse activity is filtrates from water-soaked tissues, now exhibited rather
high IAA oxidase activities . The only exceptioa was the Rosa tissue which showed only a small amouat of activity . IAA Oxidase Activity in Homogenates When we fouad that all of the tissues "leaked" IAA oxidase into the calcium
chloride solution, we werm quite surprised as it has been very difficult to detect
IAA oxidase in tissue cultures as mentioned above . Indeed, there are only negative
reports of such activity in homogenates of tissue cultures (see ref, 3, for example),
Therefore, it seemed to be of interest see whether homgenates of some of the tissues uader iavestigstioa exhibited oxidase activity . Five grams of tissue were homogenized in 20 ml water in a YirTis homogenizer, The brei was centrifuged at
58
RBIBL~IS OF SNZYt~B BY PLAIaT TI83IIS CIILTÛR&3
No. 1
Table 5 IAA Oxidase and Phosphatase Activities Obtained from Soaking Cell Wall Preparations in 0 . 05 M Calcium Chloride
Tissue s
Phosphatase b Cell Wall Homogenates
IAA Oxidase e
2
540
2000
175
3
375
900
175
4
430
1000
175
7
1340
18,000
175
8
1200
9,500
175
9
1540
18,000
175
For explanation of the superscripts, refer to table 1 . 20, 000 x G for 20 minutes . The supernatant was made up to 25 ml aad assayed in the usual manner for IAA oxidase . Aliquots of the same supernatants were also dialyzed in the cold against several changes of distilled water for 24 hours and also aaalyzed . Table 4 summarizes the results . For ease in comparison, the data for TAA oxidase activity contained in table 3 are also included . Only one tissue homo-
genate, that of PelarQonium, exhibited any oxidase activity. The dialyzed homogenate of H-196 contained some small activity. These results confirm those of other workers in that they indicate the extremely rare occurrence of IAA oxidase activity in tissue culture homogenates . Nevertheless, we are still faced with the evidence which indicates that these tissues can release IAA oxidase in the presence of calcium ions and, indeed some of them in the presence only of water . Thus, either the enzyme (one or more components as suggested by Lipetz and Galston 3 ) is destroyed by the homogenizing procedure or, an undialyzable inhibitor or a denaturant is released or produced by
lfo . 1
ItE?E~S~ OF ~IZâ~3 HY ?LA1R ?~SSIIE CIILTtIit$S
59
the process . However, the much milder soaking procedure does not inactivate the enzyme nor release or activate inhibitors . Why tissue cultures should be so unusual with respect to homogenates is as interesting problem since homogenates of organs of intact plants frequently have high I1~A oxidase activities .
Activities Rec overed From Cell Wall Preparations . Ia order to ascertain whether the enzymes were in fact bound to the cell walls and then released bY the calcium ions, cell wall preparations of some of the tissues were tested. Five grams of tissue in 25 ml water were homogenized in a VirTis homogenizer for a full minute at top speed . This procedure ensures about 90 % disrupted cells $ . The brei was centrifuged at low speed (470 x G) a.ad the supernatant discarded . Twenty ml water were added to the residue in the tube and the contents vigorously stirred and again centrifuged at low speed. This was repeated 3 more times so that the cell wall preparations were washed 4 times . The washed residue was then suspended in 25 ml 0 .05 M CaC12 and incubated at room temperature on a reciprocal shaker, At the end of one hour, the mixture was centrifuged and the supernatant assayed for enzyme activity. Only IAA oxidase and acid phosphatase activities were determined for nix of the tissues . The cell wall preparation supernatants of the six tissues all showed maximum IAA oxidase activity (table 5). Since the reaction mixtures ~~n s'n d 1 751wg IAA sad all of it waé destroyed, it is impossible to determine the relative activities of the preparations . What can be said is that all six tissues exhibited high IAA oxidaee activities . The acid phoephatase activity exhibited by these preparations is tremendously greater than that recovered from calcium filtrates in which intact tissue had been soaked. For comparison, data obtained from phosphataee assays of homogenates of these tissues are also included in table 5 . The homogenates of these six tissues contain high phosphatase activities . Thus, it would appear that relatively little of the enzyme leaks out except perhaps in the case of the three endosperm tissues (table 2). Nevertheless, the supernatants from the calcium-soaked cell
wall
prepa-
rations exhibit very high activities . It seems reasonable to assume that the cell walls bind the enzyme during the homogenizing procedure and release it in the presence of calcium ions, The same thing is probably true of IAA oxidase . The simplest interpretation of the enhanced release bY the intact tissues of certain . of the enzymes -by calcin:a ions is that gees enzymes are continously being
60
R81a1S6 ~Oh BüZII~.9 BY PL~11T TISSQB CQLTUi~3
No. 1
released by the tissues in greater or lesser amounts . Some of the enzyme is bound to the wall or, possibly the outer membrane surface and the calcium ions serve to release the enzyme from the binding sites . Japsen, et al . 13 have shown that cell wall preparations of Ay~aa coleoptilee do in fact bind enzymes . With regard to amylase and phosphatase (except for the three eadosperms and M-222), it is probable that these enzymes are not released is very great amounts by the tissue cells . Thus, if the calcium ions do indeed serve to preserve the integrity of the plasma membranes, then the enzyme activities recovered from the filtrates of the washed tissue soaked in CaC12 solution represent at least a portion of the quantity of enzyme which had previously been released by the tissue and had been bound to the cell wall . Discussion An important question, of course, is the mechanism of enzyme release by these tissues . Are they released by dead or dying sells ; are they actively secreted ; or do they diffuse out passively ? The data presented in this report do not permit any unequivocal conclusions to be drawn in this regard. Other workers 1 , 2,14~ve presented evidence which indicates that the release of amylase by tissue cultures of higher pleats is not dependent upon moribund cells . However, except for the intrinsic interest of the problem of enzyme release, the important fact is that enzymes are is some manner released from the cytoplasm into the medium upon which the tissues are grows or are bound to the outer surfaces of the cells . A tissue culture of a higher pleat has a different set of "problems to solve", teleologically speaking, than the intact plant from which it has been derived . Given minerals, atmospheric gases, and water, the intact green plant synthesizes all of its metabolites . Additionally, the vascular plants have spedalized transport systems fpr the movement of the elaborated metabolites to a]1 parts of the plant . Tissue cultures, on the other head, must be supplied not only with minerals, atmospheric gases, and water, but frequently with auxin, vitamins, and an organic nitrogen source . These substances must the taken up by the cells in one form or another and must also be transported to cells which are not directly in contact with the nutrient medium . The situation is analogous to that of a relatively massive fungal mycelium.
xo . ~
asia~sa og xxzn~s
Bi Pt exr ~iss~ cor~tfxBS
61
Uader certain circumstances, plant tissue cultures must be able to modify the substratum in order to grow . For e~tsmple, it is known that a number of tissue cultures are able to grow on media in which the sole carbon source supplied is 1,2,4,? . starch Investigation has shown that such tissue cultures do release an 1,2,4 . amylase into the medium Thus, the ability to digest starch externally is
essential for these tissues when placed on a starch medium. Recently, Straus8 has
shown that the inversion of sucrose in nutrient media by eleven different tissue cultureè is carried out by invertase bound to the cell walls of the tissues . Straus and Gerdingl2 have reported evidence which stroagly indicates that the inability of
auxin-requiring tissue cultures derived from Ephedra to grow on media in which the exogenous auxin is IAA is due to the release of IA .A oxidase by the tissues . The tissues grow very well on media containing synthetic "unnatural" auxins . Hence, it does aot seem to be inconceivable that the release of various
enzymes (by whatever means) by the tissue cultures into the medium modifies the medium in some manner and, in some cases is essential to the growth of the tissues (e, g. , when placed oa starch media) and in others prevents the growth of the tissues (as in the case of E
edra . It is tempting to speculate, although
there is no evidence on the point, that the explanation for the difficulty or impossibility of deriving tissue cultures from moat monocot species and some dicot
and gymaosperm species lies in the inability of the original expla.nt to suitably modify the artificial nutrient medium or, conversely the medium may be detrimentally modified . Summary Data are presented which indicate that tissue cultures of higher planta release enzymes into the medium upon which they are grown . Some of the enzyme released by the tissues appears to be bound to the cell wall. Release
of indoleacetic acid o~cidase (IAA oxidase) and peroxidase ie enhanced when the tissues are soaked in 0 . 05 M CaC12 . Cell wall preparations of the tissues when incubated with 0 . 05 M CaC12 release more TA A oxidase and acid phosphatase than do the intact tissues . Thus, it would appear that the cell walls bind the enzymes during the homogenizing procedure and the calcium ions probably serve to release the enzymes from the binding sites. One of the most interesting facts uncovered during this study is the demonstration of
G2
REIFA.gS OF ENZ~3 BZ PLA1rP ?I3SÜS CULTIIR$3
llo . 1
the release of IA.A oxidase by all 10 of the tissues investigated when the tissues are soaked in 0 .05 !VI CaC1 2 . It is tentatively suggested that the
release of enzymes by the tiasuee~ may serve to modify the nutrient medium to affect the growth of the tissues . References 1.
F . CONSTABEL, Pla.ata . 57, 331 (1961) .
2.
W. H . K. KARSTENS and VERA DE MEESTER-MANGER CATS, Acta Bot. Neerl . 4, 263 (1960)
3.
J. LIPETZ and A . W. GAISTON, Amer . Jour . Bot . ~j 193 (1959)
4 . L. G . NICKELL and M . K. BRAKKE, Amer . Jour . Bot . 4~1, 390 (1954) 5.
J. REINERT, H . SCHRAUDOLF and M . TAZAWA, Naturwiss . 4~, 588 (1957) .
6.
F . H . WITHAM and A. C . GENTILE, Jour. Exp. Bot . lj 188 (1960)
7.
J . STRAUS and C. D. LARDE, Amer . Jour . Bot. 41, 687 (1954) .
8.
J . STRAUS, Plant Physiol . _, 342 (1962)
9.
O. H . LOWRY, in Methods in Eazymolorxy, ~ Academic Preas, New York (1957) .
10 . S. A . GORDON and R. P. WEBER, Plant Physiol . 26, 192 (1951) . 11 . S. R . BENEDICT, Jour . Biol . Chem . 76, 457 (1928) . 12 . J . STRAUS and R . K. GERDING, Plant Physiol. 3? suppl . , xiv (1962) . 13. E . F . JANSEN, ROSIE JANG, and J. BONNER, Plant Physiol. 567 ( 1960) . 14 . M. K. BRAKKE and L. G. NICKELL, Ann . Biol. 31, 215 (1955) .