DISTRIBUTION OF NITROGEN IN THE
LEAVES OF CERTAIN SPECIES OF
THE ROSACEAE

Thesis Icr the Degree of M. S.
MICHIGAN STATE COLLEGE
john HI Davidson
I940

 

 

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Sacfiittcd to LAB Graduste School of Tichixfin
State Colleie of AgriCJltlrc find Agpliei
Science in partial filfiltcnt of the

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IGQJiTETentS ior t1;

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Departfient of Chefiistry

1940

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For the proper functioning of an

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plant or sninsl the hemicsl and ohysical conditio: of
protoplesm mast be nain+ained tithin certain definite
limits. Violation of these n tarelly prescribed conditions
may resilt in temporary inactivity or deetn of the orgznism.
The vork of Addons (1), Robbins (46, 47), and Veilbrinn (fie)

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to

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inficet

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ingortrnce of this fact in their investiqstions

on protoplosn and afients exhibiting a tox1c action thereon.

When we realize that proteins ere s nrjor cortion of
all living protoglesn find are believed by some investigators
to constitite the grenter pert oi enzynes end by others to
be closely associated with enzynes it is Qlite spgsrent any
protein stidies m3cupy such an inoortant position in pre-
sent dsy biological resetrch.

Siccessfil epolicatiqn of anesthetics, disinfectan s,
finsicides and insecticifles may quite lrrrely he attribited

to their action on these two vitsl liie systems---proto-

‘ 1

plnsm eno enzymes. Etncroft one Ricfiter (5) studying the

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effects of narcotics ugon celldlsr colloidal systews have
scorn that anesthesia is s reversible coaguletion while
disinfection is an irreversiole coegllstion o: the colloid-

f-s .-

al systen titdin the cell. Also, qcncroit and nitzle (4)

studyino the proteins in toe sensitive plent, *inoss plaice,

 

in relation to anesthesie found that some of the proteins

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study of oroteins enu toeir oroocrties is essential for e

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better inderstendine of liie and desti. with txie thougdt

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in mine a sticy oi the grotcine in Eontmorency cherry

leaves (Prinus cerssus) spreied with Seve ml of the connon

 

fun;101des was the notivctin; des re oeoine tiis investiga-

tion. The scarcity of investigationel vork on proteins in

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the leaves of our comnon fruit trees end tie

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vorxsble method for their isolation nreventeo sccoeplishnent

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of tie oesireo re

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. Inst: 0, a going Or an exolorator¢

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netire res undertaken in tie hope thet by studying the
nitrogen d stribution in the cherry leaves by e method
similar to thet folloved by Osborne, Hekemnn, anc Leaven-
worth (43) some heloful information nigit he crined. The

resilts onteined fortiiied by a review of

l

on the subject of nitrojen ;istributicn in leaves of nor-
mal and ebnornel glonts, pertiCilerly those of the Rosrceoe
finily should aid in 8 better interpretstion of iuture

reserrcn on tiis problem.

TEISTORICAL

Tne historicsl development of ”ltrOTBHOHS frecti ns
found in leaves nsy best be folloved in tvo sections. (1)
A chronological developnent of the contrih tions oi tie
more important investijstors followed by (2) the develop-
ment of the netnods employed diring their investigations.
lnesnucn es proteins are nore concentrated in seeds of
plents most 01 tne early torn on tiis surject has been
linited to seed proteins.

I. Investiqstors And Their Contributions.

Roulle (49) in 1775 announced thst the relatinOJS
metter, protein, which up to thet tine vas knoun to exist
only in the seeds of vheat, sss slso present in other osrts
of various plents. Leter he isolated a proteinsceous

prodict from the juice of nenlock. In 1729 Foxrcroy (93)

\
gave an extensive account of he oecurence of coesulsble

protein in the juice of vsrious parts oi many plsnts,

and described tne net*oc bv riicn he ohteined HTGQ‘TTtiOHS

A.

of that he supposed to he pure olent elbunin.
After 1805 no more attention was osid to proteins of

A

green plants due to Einnof's (20) investisrti ns of the
seeds of cereals. The relstive ease ritn edict grotcins

could he obtsined iron seeds enphssized the difficulties
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0i their extrpction from ereen plants vitn the result tn:t

over 8 century classed tefore furthe work on leaf proteins

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spilech lcvv=u Bcc use Di tTe esee in vhicd the nitrogen

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Soluble 1n meter 43.85
93} alcohol 3.0%
0.3? alcoholic VaOH 39.33
0.3} cqaeo1s H203 7.03
Extracted residue 5.3j

As fleet active groteine were elite solahle in QQlBOlS

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:li tPe lOV'fifure in this ecee ind ceted that fiost of

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reter tre tnelt. u.»a tLe 001101o 1 co tents ol tle eque-

ous extr”ct were DTSClgitTtei hf rlcohol the chief con-
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stltleuos Vere lOlNu to De grotern la conoln.tlon Vltl a

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hottling in leaves nede sooe interestin: oos;rvetione.
Ye conclldei tfift ineewuca es toe chloroolret res ore

birtholece of or

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intiwstely rel ted. "2e fifnres melov on Coleus leévee
suhetentizte tzie etetefiedt.
Grefis 7 oer 100 3%. dry veiqht
Green leaf 3.55
Tottled Reef A 1.39
Tottled Leaf ? 1.14
Results of nitrofien anelysee on letvee in diflerent

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b;u ETCuQLN filtru an. Q. i. SlluC Ol TVgih grotglp LE-

. 1-— -rx . ~4‘ '. rt- ,L ~-". *' 9"“ fl»- . ~ n r- .
grit tion rt fil: u ..e;: .:8 ver. little e;nn”e in 40st

" -‘ -. +‘J- .1. .1‘ 4.‘ - , w . - J..- ‘.
OI lie CWISVIVAJWF‘ Oi o :, ”."t~:‘ 331.1119 (1.. “.v—m. 4.
rm '

VJ.
:5

. -w. “-1.— ' . n - _ .' . -..- ‘ - _
lure yzs an QCClgllublOfl oi ”u 1~r nitro-wn lete

+2 ~ ~ . - . a- .. ‘ 1 - -
t.e ere 1 93.0.,, lo .t1-|1 r t“e L807“.\:l5171 of

k)
0
(+
0‘
H
l
I)
F1
)J
e
,—
C;
, I
(I)
H
J
\l
I
,_
F.
\J
.J
a
( ‘
,_)
L.
‘s
W
l 1
s .1
r.)
|>-"

\ . ~ ~ 1- ‘ w : ’- g‘ a .. —. w, . 4‘ i
increcse lfl non—grate!“ iitro e“ v in c corrueno Ll ‘
I J- ‘ - ’ -
QGCICFSB in V1; ‘r t_1n nitro“, . ‘ree no oei' $79 ole

'.»\ q T ‘ “I “ 5 "i qr ': I: 4" J' ' 1 ’ “ .
lioexcts 0i yiotein ligetion, noxevei, it

-- RV. ~ .- 1‘1. —. ‘ 1-, («dL . <, - ‘ (.1 q. ’- ‘-- c»... - :1 ..
res to 10 to Olnhbh e0 tngt 1t nee grooloig renoven iv

.1

aspereeine which even in high concentra-

SD

tions was innocuous to plants. In norne- plants asper

gine did not eccunulet which indicated that it might be

q

used as a trenslocetory suhstence. Chibnell also noticed

an inter sting difference between the leaf rO‘ein meta-

(J.

P
bolism in normfil end etnormel rinner Jean plents, the
abnornelity being drOieht. The leaves from p rnts
affected by drougit when detached and xleced in meter

eaoxsd considerehle increase in non-protein nitrofen,

O'er fiftv per cent of the increas beinr die to afiide

3 .L‘-‘,-

r'. . -L . .' r4.-.” , .3 -> - .1. -,.,: '.,. , ‘3 .,..,
.L'DJJ'IU. u.u‘ J fll..i‘f‘--, L-Ccr 0031;.-. 7,8 QELGClfim l L 1.. 3?;

.-.1,---... J,‘ 4 -‘...J— i..- ' ' * ° ' '
COdfilc : filtflbtt solltion. Yllefl J‘"“-"""’.--.'~'.‘L‘.’ LQlT? ’77

no“ . ..l A ‘ : w..- .,- .7. - ~ - -
t,e ileite a?» ceneivegslie nitrite end in ert in re—

' .. . J- . -’ . 4 . ..fi‘ . "‘ ' ‘
alone a trace oi fmwonie. Aiter :ortv-eiodt h Hr? tiere
. ,
.. . ., w, ,. . r .V (.1 1 . : - .2 T .~ '
mes note ” Jni rod lees nitrites miti an occeeionel
Prvetr].fiy ficwrr'tw.-o nnw~~-w- v. m + ..u L - ~v
.1 U - (— ' Jt \lt.’ \) ._ -\l l ‘. A]. ,L L .116:\/ L17 IO 1r (.1, S
hrfi elrwneJ tW<va vrg "‘pH 15 ~5tv~+ r it‘ : v~~
-LA at. _.. ‘L’ D s- 4. 1‘ \J O 5 A ‘J .. "./ k4 J’l-L ._ .L De, C l.L J'u‘l\t ( )leq
.,.s . Q. + ‘. n '. :7“ '. - '
and a fireet incretse in Ffllfl Pelee. SlfiiltsweOISly wit
4‘“; w: m f‘ M: I" . 1 .' “'. o
tiis egoe r nce oi smino PClQS stnrcq vec his ooerrin“
L s -
A: " L. y r‘ 1‘ ~ "(‘1 4-1‘ A. ~ , : "f \ a - " '- ‘.
and tne Cilorool -te oi tumzvfllfillpr leevcc 1818 lecowing

greener.

5-»
I.)
H
(.g
M
H. 5

-O+tlfl““31, Scidlz, and Lepkovsky (73)

.

.v.,. .. ‘. 1‘. "5 , ' . fi.‘ : _,° .
CO”l_-c.l ec 9 11m: 11's ezztr ct ion metros. initi

O.
H.
H
(D
0
cf-
fl)
cf-
(‘1
H

extrectioi on berterry and 31;: beet leaves. The redi-
rzin: nient extrrcted 18 to 20¢ more

alkali, and rcid with the following results:

Wter soli‘ole 72* 1.397,?

Alkali solitle F. 0.18fi

ACld eollhle V 0.75}

Totfl 011016 “ 1.753
These data shoved t53t only 3 efiell portion of the er‘o
“rotein of the cell feilef to dissolve in rater. Even

v -- s h ' f «r&m~-L .“ ,1 1 -’ ‘ i.
tiis rather brqSth RCld tre;t;eit leit ceoit 40) oi tie
. ‘3 w n . ' n ' . ‘ -.. ‘5 - .« . ~‘, f. 1 .r ‘
cell nitroaen 11 tle tieeie. ?iad t; 9, tie conciieion
~ ~': :--r--. -- a - ' r - . .-_ 1 .:. ‘~ "
was Jr:“n t“:t t'e ieeiiial noitioq id COfifiCSS e. oi nitr.

fien01e coietiiueite enclosed E" celluloee T?t€fi?l en

l/

I
J

(I)
H
v w
._ r-
P
1
(I
r"
i
H
C.)
(“I
I
)
cf
[—1.
..J
(k
)
L)
r_J
H
O

‘ww‘rynec no?
L. .A g, V ‘.. «V 1/ x,. .

W
1‘)
H.
(D
’7
F.1-
L.)
H
\J
n
C)
A
H
\L
O
“I
F’-
_)
(D
W
(.:.
a.
(D

colloidél pr eig-

- + r 1- .‘ p ._ fl, -. 1.. " ,_ _ . n .r.“ .. ° e, ,,. '- ' 1‘. fl ‘1 .,
1e. Li erJ Lreel ‘or- e glen,» lolloii-, Qiiua;il'e

J.

I

‘~ ~ . (or . . L“ L. ‘4 .~ v-~— A
100C»ij 03331: tel till SJWP prostteuic r34; .nic- Cun-
t: ;1ec .31 r""1ic czatgurinae.

-r J-‘- “ I a ' ‘5‘ ~ «A 4-‘« A - .J- fi-J-w

Defies (5?) ioiflc t u x ‘1 ere The ooiorbzniif

..1

V 1 ~ V‘ J..- -V .L‘ .' ' w .t ~ 4. ' 'r ‘
~V r) "\ NV)? ~ ‘Y‘I. .‘ 0' 1w_ .~ . " ‘ 1 ‘fi r9 ‘ V . --,-(
f 3 C'T I‘M-3i LU. (1:4. , {1 6’ u)“. ..A. ..1 -8? S La. nc‘lILJ. -L. T .1 \.1 t 1x.) 1 r. V \39 n flat
I‘ a (1"sz r“*-‘ c: C‘ )V) \1" ’ ”‘fi ) (“'3 ‘31 "PM "nf‘q ~\v‘ 4-; ‘ (\‘m
I i. ' .L -' ‘ ~U k . I J- 0.. ' L .C -’ ‘ fl .1. L 5 " .. x 'v - x ’ ’_L C) L1\4.L . L: .-
A 5 u a. V

‘ ' +‘ .. t1- 7 .. -‘ . - T ' J. ‘ r1 ‘ - -‘i

/‘ C" I“ “K‘f‘ I‘V‘ ‘ "Y‘f‘ r‘ " ‘ \r A\1Ar __ #3 a '3 -\
t, e ‘18 r- u LV... 1 .."..J )1.“ -.~_ ‘) C'-.C .4. r bp' 0 11/ -.1. LIVI

\.
(3
F“
'0

’\ _< '\ J— ,‘ I q .s I“ v« —‘ -v-. " rm 1‘, . -9 -. w .v - O 3" ~' ‘ ‘\ f‘“‘ ”-

un tin Oo-nf Lame view C rwan;a:n,e FEE-TweS v€C<;z re-
‘— «‘a ~1.— F‘ , . F- H". x H J-‘A -‘§ - «‘14 I. "H ‘. 1 4‘"

0.1060. 13 ‘rl‘O I," .. BILDDSITC uO C~-,:‘:‘.’l€:S-\-- J ..Cie OCCJliEC ..ilCu

, 4.- ~— ‘ . . ‘ ..t -- ,4. ,.. x x.- r-1‘\ 1‘} .L‘

QTOUCOldS e :10 witi lllvlcr recre~ee in leeOlfCr9o€S

eeedwilctee V1

1
n}
_)
‘1
, I
O
g...)
F].
‘1

evential injury to the leef under conditiahe 0i xtrefie

‘5
v

‘l .. ‘ ,‘ .-1" f .' H‘ -
cercongcrrte eeiicieicg.

3

)
3
D
J

. ._.,_ '. r‘ 1 ,_ n ~_ ~-.~~ 4. LN,"
Tho we 11 13?? (53-5/) “fie a rwt er co TISUQ 3b4L‘

1 ‘R ‘\ 1 “xx. ~~ ‘- .‘—x *f . -. . “. r: -< v‘ ' v ‘ " \
oi tie nitr3,e1 ieezunliei ll tie aiéle (liris "ll€;.

 

*. L
- ‘ ‘ j . ‘ ' 1" I ‘F x ‘ ‘5 ‘~ 2 _r‘ -‘\ 'L r : \ 1 ' - ~I " ‘ r\' 1 2-
fie etiaiea pie iueulinle 16?" greeei.e airin: tie e'r_d

: --- ,- . -. J. ' . ,. J. .L : -. n W, i, ' _ .- -' - _,~ ,. .

neriod oi Lid far ?el©fl, e» V- e Oi lrl wit retixe r31
. a 'q _- . o ‘A ”‘ .—. ‘ J. r» I“ ‘- _-. ‘ + .f n J. - - -7, s-A‘ q” ‘-

ang dirinr aeoliai». ieuyroaic 'CulVlu . _roi tie con-

,-1 . \--\ _rl n Lfifl‘r‘."‘ "I {1 tw: A_7\ Le; ‘C
3 ‘.'Il'.l‘| 3.1. 111’-'...‘J v11 4.; -(4 ;/.L\J‘.J .L .~J

— - -:.”: -'\. .:. 4.x, \ ' .. ,‘
*le”e V7? 8310 ji¢5-' “Fti~” t1 eoflcli e A i ' e1” 7;

' J. i 0 1 V- r o r I w, .A _- r‘
tlrb liutle ii ”1; wl-lltlul.0 c :(“e id t‘v n'twre oi
1“ . “ q 1‘ “if. ..‘~ . V“ ‘ -~ - -‘
t.e iuayil le leri ‘Tutclfl H—L t'-;< 31f?” c1r71" 1t“

r)
(1)
’11
(
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O
C
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‘

4' A w ' ”J. u “ Vs, ‘« - a a ‘ - ‘-
I‘Cl‘ol )1;Cll;) h‘Cv‘ cell t O J. 1?”)1 1‘16 )I‘Ooizl'f‘ ’ ' " r-o-
‘A-v‘. ‘ ‘ - ‘ ‘ I-‘« n —4 - a q n - ..,-

“ (r'+e7 tjic- .oiic ”rivet file.” Crier Lce ‘re ole co

'1' . J- 4" '- vfl" r‘ L ' L‘ ‘ ‘ 1‘1 1 v-v . . r‘nr- r - a ' ‘
qioouio5ui.e r ulufi cw'i ni'th-bl. c‘iiere.cee ll tie

ions nitroien frictions tHrOiTEOAt s yeor's cycle. Durinfi

‘ 0

this study no reletioHSQig COwld be citcovered Bet een

Q

either; 1xeegerctirc:cnésflinskine, 'z'iriinfell or'liruidity,

.1.

LV.‘ :.-...-, r» ‘
re Co er L.yort~Hb ee-

. .- _' 4_ . I
w . ~, Y‘ M". ‘Hr’
;: .._]. - U... 3 11.1. J L L

(‘3
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P. '-- ‘~ 4": 1" . ' ‘L " . 4- , Kc, - ‘ 1 r1 n r‘ v . \ -"
gen ecu tie 'reec niura en tSQQEK to Jeff inverselx re

V

d
(D
(+-
.-

d-
‘3
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{‘1‘
L P-
(1‘-
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.. . . i .4. .:. ' .:. ,..\
r3181, nOfl-pro 56;!1 n1 LIFO -(‘Jn

m'll's (1.) con-

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Flt

. J- . . ‘ya . . -., . M . -. . .._ ‘ f 7 -': 4““ r‘ 1‘ 2: ‘
t‘Ilol_Y1 {41334}; 5" 1’3 Tilt."‘- CV1 1.57.” COi'l'JCC‘tCJCL s ..LU J. iJI'DIJLlJ'I
‘. -: r.—~ 1a.- 7 . ~-\. n r- . 1‘- x 4‘ ‘. ‘1'.'4 \r— :r‘
sYntiecis FLO tie ”res ' nitiogen \lbfl crouein oei CLm'
o 1 n ‘L- J“ ~ i '~ ..~ 7- x Q‘- r‘ 1 W I ‘ 'r M
tlgq; (x) elf» proteins were tr HSBOlva to firOPlfls

tertc in the form of RTiWO ecibe

‘0 .
t’.‘
L3
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(—*.
cf-
r—
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4‘. ~ rm. , .-‘4. :.-\ .. 4. M ..1 '4. “A ‘
Roles in t1e lie" lo».e: (a, tree, 8011 Le micro en 15

only 7 to 103 throxfidoqt the ofol,; end (9) eefioni: nit-

\l

T)”.

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at.
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J81.

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111-91 0 L!

1’1

1V I’ ‘; ’. . ‘ . 1- y“ '. f" ’s 1x ' . ‘ 'Q - T\«— ‘rJ— , ' ‘ .-.Q L .~ ~ '.~ ., ;.1
thC re.elq13’ quollule nitro‘on * olor‘cfit, o1» +LlCLy

1:7 . ‘- ‘1 ‘L ' ’1; "‘.+'1~‘ Fifi. “ Vv: 1 11v" J-qu ‘
lelheu olfigl; .lul v llols lows oi huczolqae ole 3:0-

'I

m. -‘. r- I; ‘4 -- .--V \ Y'- . 1-. -.II V I" \ \ ‘
teln alb” ,01c l ,nzglcz. SlFtlrLlnT i.ctjrs 1816 ;e

r‘ . '1 . " ‘- ‘ ‘ ‘ '. r- J" 1 r‘ -' -. 4- —~ : “ ‘ - n A ‘ r :
LelcltJ ol t1; tlc91e, n_o,.r.ll~ con” lpfih + nwlns nc
. 1 a _- ,1 fl - o A L _ 1' P J. _ .‘ J J. 3 P. ‘ T! I: ‘5 a ‘ L‘n
lfl'uithr Lu klrlnuogr be tie blemie. lo Stlcu u ole

J.‘ , l .l L _, 4.‘ . .r‘ -.1- a h ..; . ,‘ J’V-
d ;}C‘.)blc4g.bloq 04. C Lo'3.Jl-:€ 'LC -?r)tCl.LS

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g.
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the iollo?inf iirlree vere obtai“efl.

7' T" ._ "I . '_ w“ ' . . 1, ‘v ’ {'1 r’ ‘7‘ '
p; dessert Lot~l ; 1n lotrl ‘ 1n -ot l , 1n
. .—I
T.“ l‘
uxtr ct a;

Extrfct Extract
Too-protein Erotei

H
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13.5 70.35

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, _' . ".fi 4.._.‘! 4. ‘_'.;‘..
1e enloos ol Lelcr,l e la: Qlli

IN‘

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.5

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It $33 also shovn th't
e; l ,4 one soother in their efiectivcnese to extr ct

V -\ 1 .' ‘. ~ ' , '1‘ I "'. .- .IQ.‘-\r\> "57 I". ‘ -‘~~‘
't031-87lC grotelns. tale x,9 lfl a lee eqt Iltl tgb

(7)
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ilLC/'..t l u‘$-c ljr FullCollil lflQLflulgllo . in to

cell their calloif'l nrtxre yrovider in ‘devl rcfct

fieclzi For vitr‘ li:e firoceerec.

vonflller (:6 1rstr3fi (31) etlfifL“: e: l'ine
wolisn 10116 no easemtisl diiiereqce T€””Tfi.7 rr
iv C loroah“ll cefecti~3 “It“fi,f of *rrlej in} t1
Ql’lt. “fl t1€ other Kefig calorlere Frti oi V'Il

 

lelzrg~vinfi le?Ve
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COflEluef-ch v_Il

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lcyll -10 «“9 Led; r 11

f“ l ' VA 1‘

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amide (*sn'rsfiwxe
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Llflu” .wlez tlhe

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OCClrred.

+

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1 ‘ ’\ e r . . ~ J". 1“". ‘5‘.- I ‘1
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left lfl a rfc113 oven 7t @307. until CO"C*FRt 7e
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vere leei fyr t“e 51:0 t: gotrl nitrnéjn deter
tiOfiS. 163F‘CEd tests vitV di3571f1"1ihe rc'"ar
3y “srvey (if) frile to infliC'te tie ireeeflce 3
tea or nitrites in t leaves. ‘He modified x3
Gunning ( 2) meth3u v33, thcr3f01e , ed for i *

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The soluble nitrogen frictions vere oht'ined by
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until examination revealed no large lesf segments. Fore
water was then added in an attenpt to aid the separation
of the fluid from the residie by vacuum filtration.
Paper gulp on a large Euchner funnel was used as the
filtering mediim. This filtering process mes very slow
due to the viscous nature of the extract. Five or six
hours vere reeiired to obtain 500 ml. of 3 clear brovn
filtrate. Piltrntes vere obtained by tfiis netfiod usicj
vster, ether-meter and trichlorecetie scifi es tie ex-

trecting median. TGTtR for protein orecioitction such

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smo.o mma.o mH.H mo.m Hu.¢m sn\a\s = = no ea
mmo.o msa.o so.H om.m eo.mm sn\H\s a = so ma
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mmo.o esH.o 55.0 mm.a ¢¢.o¢ bm\n\m = = mm m
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Davidson

T612.015 D252 126712
Davidson

Distribution of nitrogen in
the leaves of certain specim
of the Rosaceae.

 

 

 

 

 

 

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