INVESTIGATION OF ZK-BOUND PKOTEINr IN ALFALFA UFING ZN^S
by
WARREN DUDLEY RFYNOLDS
B. S., Kensas State College
of Agricultuie end Applied Science, 19S5
A THESIS
submitted in partial fulfillmeat of the
requirements for the degree
MASTER OP SCIENCE
Department of Chemistry
KANSAS STATF COLLEGE
OF AGRiCULT'JPE AND APPLIED SCIENCE
1957
•v
TABLF OF CONTEIITS
.
INTRODUCTION
...
1
SURVEY OP LITERATTJRK
2
EXPERIMENTAL
10
Instruments Used.
..•.••. •«....*•••..
Colorlmetric Analysis for Zn
11
Scxhlet Extraction
Separation into Cellular Constituents.
11
......
Radiotracer Analysis for Zn^5 in Alfalfa
.15
19
Soxhlet Extraction
Separation into Cellular Constituents.
.10
,21
.
23
Uptake and Distribution of Zn^5
in Alfalfa
...«*.«...•
23
DISCUSSION
2k
CONCLUSIONS
31
ACKNOWLEDGMENT
33
LITERATURE CITED
3>k
INTRODUCTION
As early as 191U, the requirement of zinc as
a
trace
element for plant growth In both vegetative and forage crops
was noticed (29).
It is known that the lack of zinc in the soil
produces such outwcrd effects in the tomato plant ao chlorosis,
rosettes of small leaves, stunted growth and severe necrosis (22),
Other scientists have found that the detrimental effects upon
plant growth to be universal throughout the many different plants
that have been studied {}kf
t>,
7,
31, 30, 23).
The e:ract ntetafcolic role thtt zinc plays is not known.
It
Is generally agreed that the zinc in some T»ay is responsible for
the production of indole~3-acetic acid which is an auxin or
plant growth regulator (23).
zinc
One investigator has found that
is necessary for the synthesis of tryptophane in ths tomato
plant (17).
Another investigator has shown that the zinc is con-
cerned with the phosphate turnover in the tomato plant
(2iv).
A
third investigator fo^ond that zinc is responsible for the
aldolase activity in the clover plant (21).
There has been
relatively little work reported regarding the function of Zn in
the plant and its ultimate location in plant tissue.
The purpose
of this investigation la to determine the uptake, distribution
and final location of zinc in the alfalfa plant
(
i-iedicapo
sativa
One method of zinc analysis that can be used is the single
color dithizone method.
by the A.O.A.C.
(20).
This is the method of analysis accepted
Due to the advent of the nuclear reactor.
).
a
suitable zinc radiotracer has been made available.
Zn°^ which has
and a
'.veaV
a
half -life of 250 days and is both
beta emitter (9).
This
mal'-es
u
It is
gamma ray
possible the "tagging"
of zlnc-contelning substances and subsequent radiotracer
analysis (15) •
"
SURVEY OF LITERATURE
•
-
-
In 1925, Somi.ier and Llpman stuciied the effect of the lack
of zinc in the sunflower (iv.arf var«
and barley plants (29).
)
Photographs accompanying the text showed the effect of lack of
zinc,
3oth types of plants grown Ir Zn-deficlent solution were
stvmted in growth and had poor root systems.
They stated that
the effects were manifest in the etrly stages of growth showing
the need for zinc from the very atartt
Somjuei
,
in e later paper,
showed that the buckwheat plants developed the same effects (30).
The Viindaor beans and the red kidney beans responded in a slightly
different manner.
In both of these cases, there was no visible
difference between plants grown with and without zinc until the
flowering stage was reached.
At this stage, there was
a
sudden
and rapid abscission of leaves,
Hoapland, Chandler and Fibbard have shown that the "Littleleaf" aiseai^e or rosette of fruit tree leaves was caiised by
lack of zinc (13).
a
They gave further evidence on the removal
of such effects from the orchard trees and several plants which
they had investigated.
Chandler, in
a
later paper, gave strong
evidence of the necessity of zinc In higher plants (8).
He
discussed many of the 59 references to the nort
doiie
In detecting
2lnc dericlent synptonui and subsequent removal of the deleterious
effects.
He also advanced the Idea that elnc Is a catalytle
agent In some essential carbohydrate reaction*
Stout and Arnon found that poor root development and
stunted growth in the tomato plant could be attributed to the
lack of zinc in the nutrient solution (31).
They also found the
deficiency symptoms to appear quite early In the growth of the
plant.
Reed (23) did a very careful cytologlcal study of vegetative
buds of the peach and apricot trees which were »lnc»def Icient,
The principal effects of sine deficiency were shown by several
v^ell-deflned biological changes*
He also pointed out that there
was an increase in the aocumulation of
as phloroglueinol, in the cell.
e
phenolic substance, such
He asde the valid assumption
that the trees aioblllxed their scanty supplies of zinc and trans*
ported It to the centers of maximum activity*
Several worVers have Investigated the kidney bean (7),
They found effects produced from lack of zinc similar to those
reported earlier in the tomato plants.
Ina recent investigation
Viets, et sL, (34) observed the deficiency symptoms in field»grown
Red Mexican beane.
other plants.
The symptoms were similar to thoae found in
The symptoms were general chlorosis, aubsequent
stsges of interveinal chlorosis, formation of brown spots in leaf
mesophyll, absciasion of flowers, curvature of leaves, and
necrosis.
Hewitt and -^olle- Jones (11) studied the legumes for the
effects of zinc and copper deficiencies.
They found that
alfalfa wao Icso affected by lacV of zinc than copper deficiency.
Skoog (28) concluded that Zn hcs an important function in
maintaining the normal susin concentration in the tissues.
His
experiiaents carried out en Zn-doflciont tomato plents under
different environmental conditions pave different auxin concentrations.
Prom his experiments, he stated that Zn is not
principally reculred for the synthesis of auxin.
indole-3-acetic acid (lAA),
it was inactivated more
v^as
rapidly than by control plants.
was shown by the retarding of stem p-rowth.
had
a
When an auxin,
added to Zn-deflcient plants,
This
These plants also
higher oxidation capacity than the control plants.
The
increased capac5tlep for oxidation and auxin Inactivatlon are
possibly correlated.
Addition of
plents caused an increase within
a
2i|.
Emiall
amount of zinc to the
hours in auxin production
end elongation of the stem took piece soon after,
Tsui (33) has grown tomato plants in water culture solutions
with and without zinc.
Fe found that the free auxin content of
the control plants gradually increased with the age of the plants.
The auxin decrease in the Zn-deflcient plants occurred before
there was any cecrease in growth or any symptoms were noticeable.
Whien rinc
was added to the nutrient solution the free auxin and
the enzyme digestible bound auxin increased within two days,
whereas the bound auxins released with acid and alkali showed no
increase until four days after Zn addition.
Synthotio
1-tryptophane was converted to an active growth substance to
5
the same extent by leef-discs from control and affected plants,
Zn-deficient plants contained significantly smaller amounts of
tryptophane than the control, even before there uere visible
symptoms of Zn-def iclency.
there
Three days after the addition of Zn,
noticeable increase in tryptophane content.
Y<as a
He
finally concluded that Zn is required directly for the synthesis
of auxins.
Nason (17) found from his experiments vvlth
fungi that
a
specie of
a
relationship exists between zinc and the enzyme which
converts indole and serine to tryptophane.
Cell-free enzyme
extracts from organisms grown in Zn-deficient media have shown
considerable less tryptophane production than controls.
The translocation of auxin might give a lead as to where
Skoog (2?) investigated the auxin trans-
the auxin was formed,
location in both the tomato and squash plants.
He found that
when lAA (auxin) was applied externally to the roots that it was
transported up through the xylem.
It moves laterally from the
xylem into surrounding tissues of the stem and leaves and is
then re-exported by the normal polar transport.
that the auxin may act
in two ways.
He concluded
First, high concentrations
may be absorbed and act directly on these tissues.
low concentrations (ca 3 x lO'^"
rag
Second, very
per liter) are not absorbed
into the aerial portions but may act indirectly through the
influence of the roots.
If Zn is bound
to some auxin, a method of extraction vvlll
have to be found in order to Identify this constituent,
and Skoog (32) investigated one specie of fungi.
Thimann
They have found
that the auxin can be removed by extraction with ether for three
Other solvents, such as chloroform, ethyl alcohol, and
months.
water, are less satisfactory.
The extraction process cannot be
quickened by steaming, boiling at different pH values, extraction
They foimd that water
in the soxhlet apparatus or grinding.
isas
essential for the hydrolytic liberation of auxin from tissue.
High temperature stops the process which sets free extractable
auxin.
Low aiixin solubility was attributed to slow process at
work in tissue which sets auxin free.
Since it was noted earlier that Zn has a direct influence
on tryptophane content, it was thought necessary to refer to a
reference on tryptophane production,
have shown that there is
a
Virtanen end Laine
{]>S)
definite increase in the tryptophane
content from early stages of growth to maturity in both the pea
and red clover plants.
The content reaches a maximum at an early
stage of growth, before blooming.
At this stage the per cent of
tryptophane-N in both species is double that of their respective
seeds.
The per cent content of tryptophane then falls rapidly
until the start of blooming, at which time it is only slightly
greater than in the seeds.
Reed
(21^.)
through his experiments on the tomato plant, found.
that Zn-deficient plants contain greater amounts of Inorganic
phosphate and phenol oxidase than in the controls,
lie
has shown
that affected plants contained more reducing sugars, but slightly
less sucrose and starch than those of healthy plants.
This indi-
cated that one of the essential enzyme systems failed or was
blocked, presumably by stoppage of the hexokinase catalyzed
".
.^i
^"'j
glucose to glucose-6-phoephate reectlon.
He also stated that
affected plant stems were richer in phosphatase and poorer in
hydrogenase.
He discussed
a
reference by Warburg and Christian
who worked with the yeast enzyme, zymohexase (aldolase).
enzyme is
a
This
metal protein containing 1 gram atom of Cu and 1 gram
atom of Zn per 3
3c
10" grams protein.
The enzyme may be blocked
by the formation of a complex with cystein but can be reactivated
by Zn, Cu, Fe, and Co.
Zn reactivates it regardless of the oxy-
gen tension and its presence is of prime importance in the sugar
metabolism of the cell.
Reed stated that the data now at hand
are sufficient to justify the conclusion that the presence of
Zn is necessary for the production end consumption of carbohydrates.
'
•»
Qulnlan-Vvatson (21) has examined Cu- and Zn-deficient oat
plants for aldolase activity,
Vshile the
aldolase activity in
Cu-deficient plants did not differ from the controls, the aldolase activity in Zn-deficient plants was markedly decreased.
He
stated that the breakdown of normal carbohydrate metabolism is
due to the decreased aldolase activity, which catalyzes the
reversible reaction between hexose phosphate and triosephosphate.
This last statement is in agreement with the earlier reference
of Vtarburg and Christian,
Jagannathan and Singh
(II4.)
have shov»n that Zn is necessary
for the activation of aldolase in fiingus.
The enzyme
iivas
inhi-
bited by rjetal chelating agents as EDTA, e, a'-dipyrldyl and
o-phenanthroline but the inhibition was reversed by addition of
6
Zn.
Nason, et al.
,
(18) worked with cell-free eytracts of the
apical leaflets of the tomato.
They investigated, separately,
the effect of Zn, Cu, Mn, Fe, Mo and B deficiencies on the
enzymes.
They found that Zn-deficient plants contained greater
amounts of poly^^henol oriuase, ascorbic acid oxidase, peroxidase, lactic dehydrogenase, glycolic dehydrogenase, DPNH
diaphorase but
a
less endogenous oxygen uptake.
They have
experimentally ruled out the fact that direct participation of
the metals as inhibitors of those enzymes which increase <ana©r
deficiency conditions.
Addition of the metals to the corre-
sponding deficient extracts has given no change in enzyme
activities.
However, addition of missing ions to corresponding
nutrient solutions resulted in varied responses of the already
modified enzyme patterns.
Arnon, et al.,
(1) have
analyzed the chloroplast fragments
and whole ground leaves of the sugar beet and chard plants for
B,
Mn, Cu, Zn and Mo.
They found that Mn, Zn, and Mo
v.ere
not
concentrated in the chloroplast (grana) fragments but that Fe
and Cu were.
Foster and Denison (10) studied two acid-producing fungi
as tc Zn-metabolism.
They concluded that Zn is essential for
the synthesis of pyruvic carboxylase even though indications are
that the metal is not part of the enzyme.
They stated that it is
possible under conditions of Zn-def iciency an inhibitor is formed,
Some work has been done with Zn^^ distribution in plants.
Bergh (3) sprayed ZnSO^ containing
Zn^i'
arouna the soil of
W '-v^(V -T"**-
10 plants of
"
'
Pi sum sativum var. medullare (pea plant)." Thirty-
six days later the plants were harvested and the amounts of Zn65
in the plants were determined,
l.Oii
soil was recovered as follows:
pea 0.19 per cent, shell 0.16
per cent of ln^5 added to
per cent, flower 0,01 per cent, blade 0,2? per cent, stem 0,11
per cent, root 0,30 per cent,
Biddulph
(I4.)
discussed several
references on translocation of radioactive mineral nutrients in
bean plants.
The accompanying radioautograms of leaves end roots
showed that Zn^S is deposited mainly in the veins and active
growing portions.
He attributes this deposition to the precipi-
tation of Zn as Zn-phosphorus complex.
He pointed out that as
the t concentration increased, the amount of Zn^5 deposited in
the leaves also increased.
Addition of
1
ppm of Fe reversed this
increase of Zn^3 deposition due to the formation of the more
insoluble ferric phosphate complexes.
When Zn63 was injected
into the veins of the leaflets some Zn^^ was translocated to the
roots.
of Zn 3,
This showed that there is
Wallihan, et si,
a
dovinward movement to the roots
(36) have shown,
through their experi-
ments with citrus trees, that absorption and translocation of
Zno5 occurred more rapidly in young leaves than old
leaves.
They
found no difference in the ultimate distribution of Zn in the
plant whether it was absorbed through the roots or throuf^h the
leaves.
Although, Zn is more rapidly absorbed when applied near
the center of the leaf than when applied near the margin.
radioautograms have indicated that
part in the veins of the leaves.
found earlier by Biddulph (i^.).
Zn^'^
Their
is deposited for the most
This is In agreement with that
10
Bean (2) attached the problem of Zn metabolism on the basis
that lack of Zn blocked one of the steps in the nitrate protein
buildup.
He found that in Zn-deficient tomato plants the protein
nitrogen decreased more than in controls.
He went further and
stated that this was due to the retarding of nitrate absorption
He has found an increase in soluble organic
and redaction*
nitrogen in Zn-deficient tomato plants.
10i|.
He discusses manj of the
references listed.
SXPERIMENTAL
>•"'••"•
'
-
Instruments Used
.
The instrument used in the colorimetric analysis of Zn was
the "Evelyn" photoelectric colorimeter.
mum transmittance at
5i+0
A light filter with maxi-
millimicrons was used in all transmission
'
readings,
The NMC model PC-3 alpha-be ta-garama proportional counter was
used in all radioactive se nple counting.
The gas mixture used
vas P-10 (10 per cent methane, 90 per cent argon).
All operations
were performed as directed by the operating manual (19).
obtain the operating voltage for beta-gamma counting,
curve of voltage vs. counting rate was made.
voltage was found to be
the following page.
ITisO
a
To
standard
The operating
The standard curve is on
volts.
Instrument variations with time of each
counting were overcome by counting
a
National Bureau of Standards
beta-emission standard (no. 3213, Ra D and E) with each group of
samples.
All samples coxmted at subsequent times were corrected
11
16
Voltage x 100
Fig. 1
20
12
according to the original standard count rate of the beta
Decay of samples was not included in the correction.
standards.
The minimum detectable amount of Zn°5 is about 3 x 10"-^^ grams.
This gives
a
rate approximately three times the background.
Colorimetric Analysis For Zinc
Soxhlet Extraction .
Two different samples of alfalfa were
used for the coloriraetric zinc analysis.
Both were grown in the
same field but one was commercially dried at the aehydrator
(ca 650OC) and the other was dried at 50°G,
samples were ground to pass through
Approximately
1+0
After drying both
30 mesh screen.
grams of each alfalfa meal were placed in
individual soxhlet extractors.
order were!
a
The first five extractions in
diethyl ether, skelly solve B (hexane), 95 per cent
All solvents
ethyl alcohol, redistilled water, and 0,1 N HCl.
were distilled before using.
pyrex glassware.
Vieter was
double-distilled in all
The extractions were made for
a
period of
eight hours since previous work has shown this to be sufficient
time (26),
weighed.
The extracts were all evaporated to dryness and
The weighed extracts were then dissolved with a mini-
mum of con, HgSOj^, oxidized as far as possible with con. HNO3
and finally with 30 per cent H2O2.
Volumes of
H2S0ij.,
HNO3 and
30 per cent HgOg used were recorded.
The remaining meal was removed from the extraction thimbles
and extracted at room temperature with first, five per cent
Na2S0|i^
and then O.IN NaOH,
This was done by stirring the meal
13
Fresh
and the extractant for three hours, followed by filtering.
extracting solution was added and the extraction repeated tvsice.
Thus, total extraction time was nine hours and about 900 ml. of
The same method was repeated
solution was obtained in each case.
using
a
O.IN NaOH solution.
In the first experiment, the five
j,er
cent
Na2S0||^
extracts
were evaporated to dryness, taken up in concentrated H2£0^ and
Weighed samples of Na2S0|^ and NaOH were
oxidized as before.
These corrections for zinc were subtracted
analyzed for zinc.
after suitable blank determinations.
In the following two experiments, the five per cent Na2S0h^
fractions
\.ere
placed in dialysis tubing and dialj zed against
running v.ater for
hours.
l+d
The O.IN l^aOH extracts were neu-
tralized with con, H2S0K and also dialyzed for
i|.8
hours.
The
dialyzed extracts were evaporated to dryness and oxidized as
previously aescrlbed.
.
The remaining residues viere weighed and ashed overnight In
platinum crucibles at 6COOC.
The entire procedure for both
alfalfa meals was repeated for duplication of results.
The oxidized and ashed residues of both dehydrated and low
temperature dried alfalfa samples v-ere filtered through quantitative paper and the volume adjusted to 200 ml.
were withdrawn and analyzed for
A.O.A.C. method (20).
was determined from
a
2.n
25 ml. aliquots
according to the modified
The concentration of Zn in parts per railllcn
calibration curve relating per cent trans-
mission to concentration of Zn,
The curve is shown in Fig. 2,
Ik
100
15
All BOlda, NHi|OH, solvents and water used in the procedure «ere
Zn deteralnations were run on the oxidising reagents
distilled.
and the blanks vera subtree ted fron the value obtained for each
extract*
The amount of 2n in the extracts are shown in Tables
1 and 2,
Ihipllcate two gram sataplea of both types of alfalfa msal
were ashed and analyred for
asthod (20),
2ja
according to the modified A.O.A.C,
The number of ppm of Zn In these saratles were multi-
pllad respsotively by the total weight extracted to arrive at the
total Zn,
In the case of dehydrated alfalfa, 87,66 per cent of
the Zn was obtained while
8^,7^4.
per cent was obtained in the oase
of low temparature dried alfalfa.
The sequence of extractions v^ere repeated on both types of
alfalfa meal at a lower extracting temperature,
Thie was done by
reducing the pressure in the extractors so that the meal teraperature during any extraction did not rise above 60OC,
stiver
The ethyl
bolls at a low enough temperature that reducing the pressure
during the extraction was not neoessary.
KagSOij^
Both the five per cent
and O.IN HaOH extractions were carried out as before.
fha extraction procedure was repeated again on both types
of alfalfa meal to provide frectlons for Kjeldehl nitrogen deter-
minations.
These results end the results of the Zn determinations
are included in Tables 1 and 2.
The total Kjeldahl nitrogen found
in the dehydrated alfalfa was 3,26 per cent while the low temperature dried alfalfa conteineu 3*3^ per cent*
Separation into Cellular Constituents .
A different type of
extraction procedure was carried out on fresh alfalfa
(i)).
Fresh
16
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18
frozen slfalfa plrnts (shove ground porticn) were ellowed to
et rcom temperature and tben Immersed In
thEP:
for
hours.
trio
blender with
e Waring-
a
s
slurry with v;eter in
minimum of vvater (ce 100 ml.
)
for four
More v.eter was added and the chopping continued for
minutes.
eight minutes.
through
beaker of ether
The ether was poured off end evaporated to dry-
The elftlfa plants were chopped to
nesE,
a
a
The green colored slurry was then filtered
strong cotton muslin cloth. The cotton cloth was tied
in the form of e beg and placed in a Carver laboratory press.
It
was pressed until the pressure remained constant at 7»000 lbs/
sq.in.
The liquid that was removed was added to the filtrate
from the muslin cloth.
The i)ressed solid material contained
unbroken cells and fragments of broken cells.
The green colored liquid was centrifuged in 250 ml, centri-
fuge bottles at 2,000 rpm for 35 minutes.
The straw colored
supernatant liquid contained the vacuolar liquids and cytoplasm.
The green residue
\\'8s
composed mainly of the chloroplasta.
The three fractions were dried, weighed and dissolved in a
minimum amoiuit of concentrated
HNO3
®^*^ -^^ i^^^
cent H2O2,
n2'^'^*li
®^^ oxldieed with concentrated
The coll wall material was dried,
weighed, and ashed overnight in platinum crucibles at 6OO0C, The
ash and residues w«re taken up in
a
minimum volume of IN HCI
and hot water and filtered through Whatman no. 2 paper into 200
ml, volumetric flasks.
The hot water rinsings were added to the
residues on the filter paper.
The volumes of the liquids in the
flasks were made to 200 ml, and 2S ml. aliquots were withdrawn
for Zn analysis.
The Zn determinations were done according to
the modified A,0,A,C. method (20),
19
The entire extraction procedure, except for ashing and
oxidations, was repeated to provide fractions for Fjeldahl nitro-
gen determinationE.
These were performed by the collec.e chemical
service laboratory.
The results of Zu determinotions of the
second extraction procedure and the nitrogen determinations are
found in Table 3.
average of
One gram samples %ere found to contain en
per cent nitrogen.
3.Cii.
Radiotracer Analysis for Zn^^ in Alfalfa
Alfalfa containins
250 days)
Zrfi^
(beta, gamaw emission, half-life of
obtained in the following way.
vias
About 300 alfalfa
seeds were planted in soil in 30 five-inch clay pots.
v;ere
placed on
weeks, about
:jO
a
sand table in a greenhouse.
of the best plants were chosen and transferred
to liquid nutrient solutions.
tained in
a
These pots
At the ond of three
The nutrient solutions were con-
pair of two-gallon capacity crockery jars with
niosonite board lids.
The covers had 15 three-ei^ths inch holes
drilled in each of them and the lids were covered with paraffin.
The alfalfa plants were removed from the clay pots and the roots
were washed free of soil.
The plants v.ere placed together in
pairs; the sterna partially wrapped with glass wool ana placed
through a hole in the Jtiasonite lids.
The crocks were filled to
within an inch of the lids Viith "Hyponex" solution.
This is a
commercial powdered plant food for growing plants in liquid
nutrients.
This was found to be more successful for growing
alfalfa plants than Hoagland's solution (12).
A Zn analysis
was performed on duplicate two-gram samples of "Fyponex" and was
20
Table 3.
Zn and protein in cellular constituents of fresh
alfalfa.
:Pe r cent of Total
1
t
Coloring
Pigments
5.00
Cell-wall
materials
Fresh eight
Extracted
(av.)
Per cent Zn of
(Total Zn Extracted
(av.)
0.67*
2.21*
V.
l?er cent Protein:
Composition: (;.bN X 6.25)
:
16.50
t
13.35
Ml. 02
I5.ii4
3.1+8
k^.2^
k2.9k
0,81
10.52
18.31
100.00
Vacuolar, cytoplasm, liquids
Chloroplasts
Totals
79.88
* average of two,
the rest are averages of three determinations.
found to contain an avera- e of 39.2 micrograms of Zn per gram.
After one week of growth in the liquid nutrient, eight of
the better plants vvere chosen for the experirnent.
plants were placed together and put through
a
Four of the
hole in
a
rubber
stopper and supported by small wooden stakes in the stopper.
The stopper, plants and support were placed on top of
bottle.
a
The same was done with the other four plants.
aide of both bottles were painted to exclude light.
were placed on a
214.
two liter
The out-
Both bottles
x 30 x 1 inch steel tray.
One mg, of Zn^5 (ZnClg in 0.9N HCl) was received from Oak
Ridge National Laboratory with an activity of
I.3I4.
mc/ml,
pre-
liminary work had shown that 0.2 ml of Zn65 solution which has
a
total activity of 0,258 mc was sufficient for two liters of
nutrient solution during the entire growth period of about 30
days.
This corresponds to 6 x 10-3 microgram of Zn^5 per gram
21
This amount was added to both bottles of nutrient
of solution.
solution and one-half hour later, three-lO microliters samples
were taken from each bottle and counted.
Leaves were clipped
from the plants at regular intervals in order to determine the
rate of uptake of Zn by alfalfa.
Soxhlet Extraction .
After 30 days, the above ground portion
of the plants i\ere removed and dried at fjOoC,
was placed inside
active alfalfa.
a
A small V^iley mill
dry box to prevent air contamination by radio-
The meal was then ground to pass through
a
30
An aliquot sample was weighed from the ground radio-
mesh screen.
active meal for total activity counting and the remainder was
placed in
a
small soxhlet extractor.
The five soxhlet extractions
were performed at atmospheric pressure.
The Inorganic salt and
base extractions were carried out as before.
The soxhlet extracts v^ere all evaporated to dryness and
oxidized with con. HNO3 and 30 per cent H2O2 under infra red
The residues were dissolved in hot H2O and the
heat lamps.
volume adjusted to 2S ml. in volumetric flasks.
The inorganic
salt and base extracts were dialyzed against running vaster for
14.8
hours, oxidized and made to the
extractions,
sarae
voliome as the soxhlet
;:
Three-25 lambda aliquot s were removed from each flask and
placed on copper planchettes.
The samples were evaporated to
dryness under infra red lamps and counted for 5 minutes in the
NMC model
i'C-3
proportional counter at 1750 volts.
The entire experiment of growing alfalfa on radioactive
nutrient, extraction and subsequent counting was repeatea.
results of both experiments are In Table
iv»
The
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Separetion into Cellular Constituentjs ^
Alfalfa grown on
liquid nutrient containing Zn°3 was eytracted in the same way as
the non-radioactive alfalfa as previously described.
The radioactive fractions were oxidised in the same manner
as before and made to volume in 25 ml. volumetric flasks.
Three-
25 lambda sajnples v;ere withdrawn, placed end dried on steel
planchettes, and counted for three minutes.
Table 5.
The results are In
In trial one, 30.28 per cent of the fresh weight was
recovered in the four fractions.
In trial two,
2i\.»lk
per cent
of the fresh weight was recovered in the four fractions.
Uptake and distribution of Zn^5 in Alfalfa .
group of alfalfa plants were grown on
Zn^i*
Each time a
containing nutrient
solution for extraction purposes, leaves were removed at regular
intervals of time.
The first leaf was taken about eight to ten
Inches above the roots about one-half hour after addition of Zn^^
Table 5»
Radiotracer analysis of cellular constituents of alfalfa
grown on Zno5 containing nutrient solution.
:ter cent of Total :vi eight :Fer cent of Total
Composition:Extract Activity (f-;rams) :Extract Activity
:
:
:
Weight
(grams)
Coloring
Pigments
0,00
0.0282
0.00
0,0587
Cell-wall
Material
l.li+36
i^..67
1.23ij.X
7,08
Vacuolar-Cytoplasm, Liouids
bl+.ll
0,6132
81.91
0,2560
Chloroplasts
11.22
O.llj.86
11.01
0.2083
100.00
2.07iil
100.00
1.6666
Totals
,
i
2k
The succeeding removal of leaves v^ere
to the nutrient solution.
from this height at regular intervals for ebout 30 days.
During
the 30 day period of aampling, nei» growth developed at the base
Leaves from new growth also were removed.
of the plant.
The leaves mere vveighed, dried at 75°C and weighed again,
51ii5r"««re
then ashed overnight in one inch diameter stainless
steel cups at 600°C, treated with dllxite Hci and the residue dried
Counting was done on NMC model FC-3 windowless
for counting.
proportional counter at 1750 volts.
6.
The curves are in Pigs, 3 to
'
^
,
.:
Radioautographs of several of the leaflets from two different
experiments were made.
This was done by placing the radioactive
leaflets directly against the emulsion of No-screen X-ray film
and held in position with
The exposure
leaflets.
ti-ue
a
weight placed on
a
nasonite board.
was computed from the activity and area of the
In the first radioautograph, the exposure time was I6
hours while in the second it was
I4.8
with standard X-ray film developer.
hours.
The film was developed
The radioautographs are in
Pigs, 7 and 8,
DISCUSSION
'
A difference in the solubility of zinc containing substances
has been found between commercial dehydrated (ca 650°C) alfalfa
end low- temperature (5>0°C) dried alfalfa as shown by soxhlet
extractions.
In the latter,
the greatest concentration of bound
zinc was in the ethyl ether extract while in the former it occurs
in the water extract.
This might be explained on the basis that
^s
N.20 -
o
100
Fig. 3.
200
_
300
400
500
Hours
Uptake of Zn^S In alfalfa plant.
The nutrient
solution contained 0.253 mc Zn65 in 2000 ml.
of solution.
too
200
300
400
500
Hours
Pig.
i|.
Uptake of Zn^S in alfalfa plant.
The nutrient
solution contained 0.0258 mc of Zn65 in 2000
ml, of nutrient solution.
26
too
Fig. 5.
200
300
Hour s
400
500
Uptake of Zn^S In alfalfa plant grown on Zn^3 containing nutrient solution. The nutrient solution
contained 0.250 mc in 2000 ml. of solution.
ISO
300
480
600
790
Hovirs
Pig. 6.
Uptake of Zn65 by new growth from alfalfa plants
grown on Zn65 containing nutrient solution. The
nutrient solution was the same as in Fig. 3.
27
Leaves removed from alfalfa plant grown on
Fi£I, 7.
Znt'5 (0,258 mc) containing nutrient solution.
Leaves
were removed 11 to Ik days after addition of ZnPB to
The small leaves are new growth.
nutrient solution.
The large leaves with only the veins showing activity
were removed 11 days after addition.
28
Leaflets removed from alfalfa plant grown on Zn^S
Pig. 8.
Time of removal from
(0.258 mc) containing nutrient solution.
plants after addition of Zn^S to nutrient solution are as follows
(left to right):
232, 89, 356; 60[^, 523, i;96 hours.
29
the solubility of the substance to %?)ich En is bound is altered
during commercial dehydration,
iToteins are substances whose
solubilities are altered when exposed to temperatures of
75°C (5).
I4.OO-
Several references have stated that Zn is bound to
certain proteins (enzymes) (37# 2kf 16).
It has been found that
there is no loss of Zn during the dialysis of Zn-protein complexes
(37, 18),
.
.
The proteins of the commercially aehydrated alfalfa are
already at least partially denatured due to the temperature of the
dehydration process.
Therefore, keeping the extraction tempera-
ture at a minifflum should have little effect on the solubility.
It has been found that a hi^h per cent of Zn was obtained in the
water extracts of the dehydrated alfalfa.
If the Zn is bound to some enzyme in the plant, then there
should be a large amount of ^n in the water and dilute acid solu-
tions extracts (p).
This was found in all but one of the extractions.
In the radiotracer analysis, the greatest concentration of Zn was
fotmd in the Na2S0i^ extract, although, 78-oC per cent of the total
Zn was found in the water, dilute acid and neutral salt extracts.
In the colorimetric analysis of the fractions from the dehydrated
alfalfa, 50-76 per cent of the Zn was found in these three extracts.
The corresponding fractions of the low-temperature dried alfalfa
are lower.
Bonner (5) stated that the plant-cell enzymes occur in the
cytoplasm.
Both the colorimetric and radiotracer analysis of the
cytoplasm (cell-liquids) fraction show the greatest concentration
30
of Zn«
The por cent 2n in this fraction vas more evident in the
radiotracer analysis than in the oolorimetric cnalysis.
possible therefore that
'In
It is
may be bound to certein ensyaea in the
oytoplaamic fluids*
A large ataount of 2n has been found in the oell->wsll aiaterial
by the colorlmetric analysis*
The corresponding fraction by the
If the Zn-bound substance is
radiotracer analysis ia such lower*
fotmd principally in the cell<»liquid, then any Za present in cell-
vail material may be aasiuoid to be due to incomplete cell«>rupture
and reooval of all cell-liquida*
Arzion, et al.»
the chard plant.
(1)
found no Zn in the chloroplast fraction of
I'his is
on alfalfa in this paper*
cent of the
'In,
in contrast with results of work reported
It has been found that about ten per
as ihonn by both uoloriiaetrlc and radiotracer
anal^'siSf vas contained in the ohloroplaats of the alfalfa plant*
From the curvee of activity vs. time, it
v.as
found that
between 65-100 hoars after addition of Zn^^ to the nutrient solution a noticeable increase of Zn^^ occurred in the matxire leaves*
This contrasts with that found
h'^
Schaff
iZ!?)
who noted f32 in
wheat leaves one hour after being added to the nutrient solution*
Tsui (33) has found that the bound auatin increased within 96 hours
after addition of Zn to the roots of Zn-deficient plants.
This
is a significant parallel in that In has been assumed to be involved
in auxin production*
The uptake curves do not indicate whether
the plant absorbs the Zn late in the metabolism cycle or if the
plant takes this much time (65 hrs*
bound cn«yme la needed*
)
-^'
before any additional Un-
31
^.
The first
New leaflets had a phenomenal uptake rate of Zn^^.
detectable uptsVe of Zn°^ wes 23 c/ra/mg at 110 hours after addition of Zn^S to nutrient solution.
At the end of 230 hours, the
activity? was 2600 c/ra/mg while the final sample contained 3i^75
The rapid and large uptake by young leaves is
c/m/mg at 33 days.
consistent with that found by Wallihan, et al.
Inspection of the radioautographs in
Pit:;.
(36)«
7
show that Zn is
Ihe leaves
contained throuf-hout most of the individual leaflets,
taken earlier in the experiment show a high concentration of Zn
in the veins.
The veins belftg the source of liquid movement from
the roots would naturally contain a higher concentration of Zn
than surrounding tissues.
It is possible that the Zn might have
been deposited in the leaf veins early in the course of the experi*
ment.
In Pig. 8, the leaflets taken at the various intervals of
time show that the Zn^i* is contained in the leaflets throughout
the experiment.
It is indicated that once the Zn^5 is deposited
in the leaves it remains as such.
both figures show
The petioles of leaflets in
heavy concentration (light areas) of Zn^3,
a
This might be due to the narrow area through which the Zn^5 must
pats, thus showing
a
high concentration in
a
small area,
CONCLUSIONS
'.
'
Prom the foregoing statements, it is concluded that the zinc
in alfalfa is bound to some non-dlalyzable substance which is
found mainly in the cell-liquid (cytoplasm) fraction.
It is also
concluded that the Zn-bound substance is more soluble in water.
32
dilute acid, and neutral salt solutions than in organic solvents.
The solubility in dilute acid is attributed to the partial
hydrolysis of the Zn-»bound substance.
The exact compound (prosthetic group) to which
i-n
is bound
and the chain of events %hich produce this compound cannot be
explained on the basis of this investigation.
It can be postu-
lated that Zn is not required or used immediately in the xaetabolism
system,
Zn has been found to be absorbed between 65 and 100 hours
after addition to the roots.
leaflets of the alfalfa plsnt.
The final location of Zn is in the
33
Acknov-ledgment Is made to Dr.
^'.
A.
schrenk for his helpfxil
criticism* snd guidance throughout this research; to Dr, R» E,
H«in for his ftsslatance In the redlotrecor analysis.
'The
author
is indebted to Dr. J. C. Fraaier who ^ave erpert advice on
growing alfalfa plants In nutrient solution.
34
.
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Videnskab. Selskab Forh. 23(16J:6fi-7, (17):6b-71.
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Some effects of Zn on several species of Gossypium L,
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1952.
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New York:
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1950,
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19i;9.
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1937.
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(11)
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1950,
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iS
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33:131-11^1.
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(li+j
Jagannthan, V., and K. Singh,
Metal activation of Aspergillus nlper aldolase.
19i:^ii.
et, Bicphy. Acta,
li,:13'^.
(15)
Kanen, M. D.
Radloectlve tracers in biology.
Press.
1951.
Bioch.
Academic
New Yorkj
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and Y<. C, Loh-Mlng.
Mediation by metal of the binding of small molecules by
76:805-8li|.
1954.
proteins,
J, Am. Cjhem, Soc.
(16)
Klotz, I, M.
(17)
liason. A,
The effect of Zn delficiency on the synthesis of tryptoSci. 112;111-112.
19i)0.
phane by neurospora extracts.
(18)
Nason, A., H, A. Olaoifturtel, and L, M, Propst.
Role of micronutrient elements in the metabolism of
Changes in oxidative enzymes constihigher plants.
I.
tution of tomato leaves deficient in micronutrient
33:1-13.
1952,
elements. Arch, of Bioch, and Biophy.
(19)
Nuclea? Measurements Corporation,
Instruction manual serial no, 16,
(20)
Official Methods of Analysis of the Association of Official
Agricultural Chemists,
Eighth
Assoc. Off. Agr, Chemists, Washington, D. C,
edition, 1008 pages,
1955.
(21)
Quinlan-Vvatson, T. A, P.
Aldolase activity in Zn-deficient plants.
1033-1031+.
1951.
Nature 16?:
(22)
Reed, H. S*
The relation of copper and zinc salts to leaf structure.
Am, J, of Rot,
26:29-33.
1939,
(23)
Reed, H. S,
Effect of Zn aeficiency on cells of ve.?etative buds.
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28:10-17. 19ij-l.
(2I4.)
{ZS)
Reed, w. S.
Effect of Zn deficiency on phosphate metabolism of the
19i+6.
tomato plant. Am. J. of Bot.
33:77B-78ii..
Schaff, J,
Unpublished M, S, thesis, Kansas State Coll,
1953*
36
(26)
Schrenk, V*. G., D. A. Nauraan, and R, E, Hein,
Preliminary report on Zn uptake in alfalfa
Kan. Agr. Exp. Sta. bulletin No. SkO, unpublished work.
(27)
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Absorption end translocation of auxin.
25:361-372.
193^.
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J,
of Bot,
Skoog, P.
Relationship between Zn end auxin in the growth of
27:939-951. 19l<.0,
Am. J. of Bot.
hljiher plants.
A. L., and C. B. Lipraan,
Evidence on the indispensable nature of Zn and E for
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1926.
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SOfloner,
(30)
Sonimer, A.
L.
Further evidence of the essential nature of Zn for the
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(31)
Stout, F. R., end D, I, Arnon.
Experimentfil methods for the study of the role of Cu,
Am, J. of
Mn, and Zn in the nutrition of hi£her plants.
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26:ll4.i+-lU9.
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(32)
Thimman, K. V,, and P. Skoog.
The extraction of aurln from plant tissues.
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27:951-960.
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The role of Zn in auxin synthesis in the tomato plants.
19LB,
Am, J. of Bot.
35:172.
(3k)
Viets, Jr., F. G. , L. C. Boavm, and C, L. Crawford.
Zn content of bean plants in relation to deficiency
195J+#
Plant Phy.
29:76-79.
symptoms and yields.
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TryptoI,
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1956.
Zn in citrus plants.
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Wood, J. G,, and P. M, Sibley.
Carbonic Anhydrase activity in plant? in relation to Zn
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B3:lll..
1950.
^>-
IHVESTIQATION OF ZN-BOUND l*nOTriNS IN ALFALFA aSIHG ZM^^
t»y
WAHREN DQDLEy REYNOLDS
B* £•, Kansas £tats College
of Agriculture end Applied Science, 1955
AN ABSTRACT OP A THESIS
submitted in partial fulfillaient of the
reqiaireaents for the degree
MASTER OF ECIFNCE
ttpaJPtoent of Chemistry
KANSAS STATE COLLMi''- /
Of AGRICULTURF AND APFLIBD SCIBTCB
.-i
1957
ThlB rosearcli Hat conductad to determine the uptake,
distribution and final locetion of Zinc in the alfalfe plant
(
Medloa/g.o
aatlve
).
Color Imetrlc end radiotracer methodi are
available for the analyela of Zn.
Two different kinds of dried alfalfa were Investigated.
One was coittwrclallj dried at the ciehydrtitor (ca
other at a low tea^erature (50°c;).
soxhlet extractions using, in order:
Botv«
6i>0*'C)
and the
were subjected to
ethyl ether, hexane, 95
per cent ethyl alcohol, redistilled i»ut«r, and O.IM Hcl.
The««
extractions were carriea out both at room tenqpereture and at
The alfalfa samples were further extracted
lower temperatures*
with first five per cent NagSO]^ and then O.IS NaOH,
from all the extractions were analyzed for
-in
The extracts
by the accepted
colorimetrle A.O»A»C, acthod*
Low temperature dried alfalfa was separated into cellular
constituents.
These oonstitueato Included ether soluble material,
cell«T;all material, cell liquids, and ohloroplasts.
was analyzed for
2-n
Each extract
by the accepted colorlmetrio A.O.A.C. method.
Several groups of alfalfa plants, which bad first been
started on soil, were trenaf erred and grown on liquid nutrient
solution containing Zn^^ (as ZnCl2).
The various groups were
grown on liquid nutrient for a period of
21;
to 33 days.
During
•aoh growth of plants, leaves were removed fro*a the plants at
regular Intervala of time
fx'otr.
one-half hour to
a
fin&l time, in
one case, of 33 days*
These leaves were weif,hed end ashed and
counted for activity,
A plot was prepared of o/m/mg vs. time.
Severfil of the
l««vao teVen
frora the
plants at various interval*
of time ^Bre radioautrographeci by plscinuj- then! against X-ray film.
The Zn^i? containing alfalfa plants sere then harvested and the
2n"^ extracted by the two prevloualy mentlonad procedures.
A difference in the solubility of 2n containing substances
has been found between cotaserclal dshydruted alfalfa and low
temperature dried alfalfa as «ho*n b> the aoxhlet eytraotions.
In the latter, the ^^reateat concentration of In was in the ethyl
ether extract while In the former it occurs in the water extracts*
In the radiotracer enalysle of low temperature dried alfalfa,
76-«0 per cent of the total
7.n
acid and neutral salt extracts.
was found in the water, dilute
This 1| oonsistent with that
found b^ the color iiae trio ana I7 sis*
Both radiotracer and colorlnetrlc analyela of tha cell
liquids (cytoplesffi) show tho greatest concentration of 2n,
It
la concluded that the £n in alfalfa is bound to aoise aoii«difcl>«able
substance which is found mainly in the oell llq.ild (cytoplasia)
fraction,
Zn has been found to be absorbed into the leaves
between 65 to 100 hours fcfter addition to the nutrient solution.
One of the final locations of Zn ia In the leaflets of the alfalfa
plant.
•
.
i>
^.