Plant Growth Substances - American Chemical Society

1 Chemistry and Physiology of Conjugates of

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Indole-3-Acetic

Acid

ROBERT S. BANDURSKI Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824 Auxins are hormones t h a t promote p l a n t growth. They occur n a t u r a l l y and, i n s t r u c t u r e , g e n e r a l l y possess a planar aromatic r i n g , an u n s u b s t i t u t e d , or e l e c t r o n e g a t i v e l y s u b s t i t u t e d p o s i t i o n ortho to a side chain o f , at l e a s t , two carbons, and w i t h a c a r boxyl group on the s i d e chain 2_). Examples of auxins are i n d o l e - 3 - a c e t i c a c i d ( 3 J , p h e n y l a c e t i c a c i d ( 4 J , and 4 - c h l o r o i n d o l e - 3 - a c e t i c a c i d (_5, 6). Knowledge o f the s t r u c t u r e o f i n d o l e - 3 - a c e t i c acid--IAA--(7_) l e d to the chemical p l a n t growth r e g u l a t o r i n d u s t r y w i t h annual a g r i c u l t u r a l savings i n Michigan alone equal to the cost o f a l l U . S . financed plant hormone research. U t i l i z a t i o n of auxins date to a n t i q u i t y , as f o r example, the use of germinating seeds to promote r o o t i n g o f c u t t i n g s ( 8 ) . The d i s c o v e r y of hormones i n roots by the P o l i s h h o r t i c u l t u r a l i s t , T e o f i l C i e s i e l s k i {9) and i n shoots by the B r i t i s h n a t u r a l i s t , Charles Darwin (lOJ was made one century ago. They observed t h a t the t i p o f the root or shoot c o n t r o l l e d growth of the t i s s u e some distance from the t i p . Thus, an " i n f l u e n c e " must have d i f f u s e d from t i p to growing r e g i o n . S i x t y years l a t e r , F.W. Went developed the "Avena curvature t e s t " f o r the " i n f l u e n c e " (3)* and Bonner developed the " s t r a i g h t growth assay" (11,). These t e c h niques plus knowledge that tryptophan could be converted to auxin i n fungal c u l t u r e s (12) and t h a t "precursors" i n seeds could be converted to auxins by a l k a l i n e h y d r o l y s i s (13^, 14_, 15_, 16_) l e d to knowledge o f the s t r u c t u r e o f IAA (7_). We s t r e s s these assays because, w h i l e l e a d i n g to the d i s covery o f IAA, they imposed s t r u c t u r e - a c t i v i t y requirements prec l u d i n g study of the IAA conjugates—and p o s s i b l y the d i s c o v e r y o f other a u x i n s . For a substance to be a c t i v e i n the assays r e q u i r e d t h a t they: 1) permeate membranes i n a cut t i s s u e s u r f a c e , 2) be transported to the growing zone, and 3) promote growth i n t h a t zone. Hopefully these three requirements may someday be s t u d i e d independently. The b i o l o g i c a l e f f e c t s o f the auxins are d i v e r s e and range from r a p i d e f f e c t s , u s u a l l y growth promotion and o c c u r r i n g w i t h i n 0-8412-0518-3/79/47-lll-001$05.00/0 © 1979 American Chemical Society Mandava; Plant Growth Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2

PLANT GROWTH SUBSTANCES

minutes, to growth d i f f e r e n t i a t i o n , obvious o n l y i n days ( 1 7 ) . How IAA can e l i c i t profound changes i n the s i z e and form o f a p l a n t i s t o t a l l y unknown. At the c e l l u l a r l e v e l i t i s known t h a t the p l a n t c e l l w a l l s must be "softened" f o r growth promotion to occur ( c f . 1 8 ) . Such e f f e c t s , however, may be concomitants o f growth and not the "primary" e f f e c t s of auxin ( 1 9 J . We suggest t h a t research d i r e c t e d towards c o r r e l a t i n g and developing b i o l o g i c a l and physiochemical assays o f the a u x i n s , s t r u c t u r a l c h a r a c t e r i z a t i o n o f auxins and auxin conjugates and s t u d i e s of what permits auxins to move to d i f f e r e n t parts o f the p l a n t and, perhaps s e l e c t i v e l y i n t o d i f f e r e n t o r g a n e l l e s , w i l l be p r o d u c t i v e approaches. Thus, I s h a l l confine my remarks t o , 1) the chemistry o f IAA conjugates, 2) the q u a n t i t a t i v e assay o f IAA and i t s conjugates, 3) the "turnover" o f the i n d o l y l i c compounds of the p l a n t , 4) an i n d i c a t i o n of how knowledge of pool s i z e and turnover permitted i d e n t i f i c a t i o n o f the seed auxin p r e c u r s o r , 5) a demonstration o f the e q u i l i b r i u m between IAA and i t s conjugates, 6) a demonstration o f the p e r t u r b a b i l i t y o f the e q u i l i b r i u m and, l a s t l y , 7) a working hypothesis concerning how a hormonal homeos t a t i c system can be attuned to the environment. The

S t r u c t u r e and Concentrations o f Indoles o f Zea mays

Figure 1 summarizes the s t r u c t u r e s and c o n c e n t r a t i o n s o f the IAA conjugates o f the kernels o f corn {Zea mays), the only p l a n t to have been s t u d i e d i n d e t a i l . This work was done by my c o l leagues Drs. Labarca, N i c h o l l s , Ueda, P i s k o r n i k and Ehmann (2025). We have not detected a p p r e c i a b l e amide l i n k e d IAA i n Zea but there are three major c l a s s e s o f e s t e r s : the IAA-zm/tf-inositols, c o n s t i t u t i n g about 15%; the I A A - r a / o - i n o s i t o i g l y c o s i d e s , about 25%; and the high molecular weight IAA 31+4 g l u c a n , about 50% o f the t o t a l IAA. Free IAA, the 2 - 0 , 4-0 and 6-0 IAA glucose e s t e r s and the ( I A A ) i n o s i t o l s comprise the remainder. The v e g e t a t i v e t i s s u e of corn contains 300 yg/kg fresh weight o f e s t e r IAA and 30 yg/kg o f free IAA (26.). A major p o r t i o n o f the e s t e r s o f the shoot i s IAA-tfz?/o-inositol ( c f . 27 and Nowacki, u n p u b l i s h e d ) . The seeds o f oats {Avena sativa) have been s t u d i e d by Dr. P e r c i v a l and shown to have 85% of t h e i r IAA e s t e r i f i e d to a g l u c o p r o t e i n ( 2 8 ) . The glucan i s o f the l i c h e n a n type having both 31+3 and 31+4 l i n k a g e s . R e c e n t l y , Ms. P. Hall i n our l a b o r a t o r y (personal communication) has i s o l a t e d I AA-mz/c-inositol from r i c e (Oryza sativa) thus showing the compound o r i g i n a t e d e a r l y i n cereal e v o l u t i o n . This completes our knowledge o f the chemistry o f the n a t u r a l l y o c c u r r i n g IAA conjugates. IAA-aspartate i s known to be formed f o l l o w i n g exogenous a p p l i c a t i o n o f IAA to p l a n t s o f Pisum sativum and was the f i r s t IAA conjugate to be s t r u c t u r a l l y chara c t e r i z e d ( 2 9 J . There are some data i n d i c a t i n g t h a t IAA-aspartate occurs n a t u r a l l y ( 3 0 ) . In a d d i t i o n , 1-0 g l u c o s y l IAA has been reported to be formed f o l l o w i n g a p p l i c a t i o n o f IAA to p l a n t s (31). n

Mandava; Plant Growth Substances ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

BANDURSKi

3

Indole-3-Acetic Acid Conjugates

D e f i n i t i v e c h a r a c t e r i z a t i o n o f the b i o s y n t h e s i z e d 1-0 glucose e s t e r has not been published although the compound has been s y n t h e s i z e d c h e m i c a l l y (32). Q u a n t i t a t i v e data on the amounts o f free IAA, e s t e r IAA and amide l i n k e d IAA have been s u p p l i e d by Ms. A. Schulze (33). Her d a t a , shown i n Table I , permit several c o n c l u s i o n s : f i r s t , a l l plants examined c o n t a i n g r e a t e r amounts o f IAA conjugates than free IAA; secondly, the c e r e a l s c o n t a i n mainly e s t e r IAA; and, t h i r d l y , legumes c o n t a i n mainly amide l i n k e d IAA. The d i s c o v e r y by Ms. Schulze o f the u b i q u i t y o f IAA-conjugates convinced us that c o v a l e n t l y bonded hormone conjugates were of metabolic importance. E s p e c i a l l y s i g n i f i c a n t was the occur rence o f IAA conjugates i n seedlings where growth r a t e i s a func t i o n o f IAA c o n c e n t r a t i o n . We concluded t h a t , i f an e q u i l i b r i u m e x i s t s between IAA and i t s conjugates, then anything t h a t s h i f t s t h a t e q u i l i b r i u m would a f f e c t growth r a t e . To f u r t h e r study t h i s system f o r hormonal homeostasis two a d d i t i o n a l t h i n g s were r e q u i r e d , one, a more convenient and s e n s i t i v e assay f o r IAA and, two, knowledge of pool s i z e and turnover rates o f the i n d o l y l i c components o f the p l a n t . Methods o f Assay B i o - a s s a y s , i n the hands o f c a r e f u l workers ( c f . 3)> have provided almost a l l o f our knowledge of p l a n t hormonal metabolism. Such assays, however, measure a c t i v i t i e s o f e x t r a c t s and are not equatable to amounts o f a chemical e n t i t y . Thus, one must r e l y upon both chemical and b i o a s s a y s . With regard to chemical assays, i t i s our contention t h a t the planar i n d o l e s t r u c t u r e and the number o f π bonding e l e c t r o n s renders IAA so unstable t h a t r e c o v e r i e s w i l l be both low and v a r i a b l e thus making i n t e r n a l standards o b l i g a t o r y ( c f . 33, 3 4 ) . We f i r s t used C - I A A - i s o t o p e d i l u t i o n assays i n 1961 ( 3 5 j and r e f i n e d the assays i n 1974 ( 3 3 ) . The o r i g i n a l assays r e q u i r e d kilogram amounts o f t i s s u e and a week to o b t a i n a s i n g l e v a l u e . More r e c e n t l y we developed two new assay procedures which permit one assay i n a day or two o f work and r e q u i r e only 10 to 50 grams o f t i s s u e (36, 3 7 ) . Our assays are l a b o r i o u s but, i n t h i s e a r l y stage o f chemical assays o f IAA, such l a b o r may be d e s i r a b l e . One method i n v o l v e s the use of 4 , 5 , 6 , 7 t e t r a d e u t e r o - I A A , r e c e n t l y synthesized by Dr. V . Magnus ( 3 6 ) . There are two advan tages to use o f t h i s as an i n t e r n a l standard: f i r s t , the deu terium i n these p o s i t i o n s i s s t a b l e to the a l k a l i n e h y d r o l y s i s we use to assay the IAA i n the conjugates, and s e c o n d l y , the pre sence o f four deuterium i n the standard moves the ions o f the standard away from the isotope c l u s t e r normally observed i n mass spectrometry owing to the n a t u r a l l y o c c u r r i n g heavy i s o t o p e s . We add d^-IAA, plus a t r a c e o f C - I A A , to the acetone i n which we homogenize the p l a n t m a t e r i a l . Then, with or without a l k a l i n e h y d r o l y s i s , depending upon whether we wish to measure free or 14

1I+



acid

)-myo-\inositol

5.6%

15.4

23.2%

15.2% 10.1

4.7%

0.8%

10.5%

PERCENT OF TOTAL

0.5

IN DRY SEED MG/KG

5-£-6-L-arabinopyranosyl-l-DL( i n d o l e - 3 - a c e t y l ) - m y o - \ nos i t o i

AMOUNT

17.6%

STRUCTURE

5-0-3-L-arabinopyranosyl-2-0( i n d o l e - 3 - a c e t y l ) - m y ο-Λnosi t o i

Indoleacetylinositoi-arabinosides

l-DL-(indole-3-acetyl

2-C-(indole-3-acetyl)-mz/o-inositoi

Indoleacetylinositols

Indole-3-acetic

COMPOUND

Ο Μ

•

Η

S d w

H

I

S

H

>


compounds

toi

itoi

Figure 1.

(indole-3-acetyl)-glucan

LOW M . W . COMPOUNDS — TOTAL 3 1 4 c e l l u l o s i c glucan with 7 t o 50 g l u c o s e u n i t s p e r IAA

2

CHOH

0.05

47.6%

52.5% 35.0

0.3%

0.2

31.2

.1%

5.4

The structure and concentration of indolylic compounds in kernels of Zea mays

6-0-(indole-3-acetyl)-D-glucopyranose

4-0-(indole-3-acetyl)-0-glucopyranose

2-0-(indole-3-acetyl)-D-glucopyranose

Tri-Ο-(i ndole-3-acetyl)-myo-inosi

Di-0-(i ndole-3-acetyl)-myo-\nos

Trace

5 - o - a - L - g a l a c t o p y r a n o s y l -2-0(indole-3-acetyl )-ra/oinositoi

n

Ci

sCιιO

§•

>

6


TABLE I Concentrations o f Free and Bound IAA i n Various P l a n t Tissues Species

IAA content

Tissue Free IAA

1

E s t e r IAA

2

Peptidyl IAA 3

yg/kg CEREALS Avena

sativa

Avena sativa Hordeum vulgare

vegetative 16 tissue 440 seed seed 40 (milled) 1703 seed 366 seed 123 seed vegetative 24 tissue 500 to seed 1000

5 7620

69 n.d.

329 2739 3198 511

-

328 71600 to 78500

60

5

Ovyza sativa Panicum miliaceum Triticum aestivum Zea mays Zea mays

-

LEGUMES Glycine max Phaseolus vulgaris Pisum sativum Pisum

sativum

seed seed vegetative tissue seed

4 20

50 30

5

5 5

35 93

5 n.d..

0 40 30 trace

905 127 no trace

524 136 43 202

OTHERS liquid endosperm Fagopyrum esculentum seed seed Helianthus annus Lycopersicum esculentum f r u i t Saccharomyces cevevisea packed cells Cocas

nucifera

5

290

5

n.d.

-

25

-

*No a l k a l i n e h y d r o l y s i s . I A A a f t e r h y d r o l y s i s with 1 Ν a l k a l i minus the free IAA. I A A a f t e r h y d r o l y s i s with 7 Ν a l k a l i minus the free and e s t e r IAA. ^Seedlings and f r u i t s are fresh weight, seeds are a i r dry and yeast c e l l s c o n t a i n 30% dry m a t t e r . A v i s u a l estimate o f IAA on a TLC p l a t e as c o l o r i m e t r y was pre cluded by contaminants. 2

3

5

Reprinted by permission o f P l a n t Physiology ( 3 3 ) .


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BANDURSKi

7


t o t a l IAA, we r e - i s o l a t e the IAA by p a r t i t i o n i n g , DEAE-sephadex, and high pressure l i q u i d chromatography. The r e s u l t a n t mixture of d^-IAA and the p l a n t d e r i v e d IAA i s methylated and then the i n d o l e n i t r o g e n i s a c y l a t e d w i t h h e p t a f l u o r o b u t y r i c anhydride. The methylheptafluorobutyryl IAA d e r i v a t i v e i s used f o r gas chromatography-selected i o n mass spectrometry (gc-sim-ms). As shown i n Figure 2 , we monitor four masses, 385 and 389, the molecular i o n f o r methylheptafluorobutyryl IAA (and i t s d a n a l o g ) , and 326 and 330, the base peaks f o r IAA and the d s t a n dard. The agreement between the r a t i o s o f d - I A A to IAA at the molecular i o n and at base peak g i v e assurance o f the v a l i ' i t y o f the assay. Is t h i s f i n a l l y an absolute assay, g i v i n g data such t h a t e r r o r i s impossible? We t h i n k i t i s c l o s e i n t h a t f o r an e r r o r to occur a compound would have t o c o f r a c t i o n a t e w i t h IAA on a DEAE and HPLC column and coemerge from the gc column and then y i e l d the same percentages o f ions at mt and at base peak as IAA. S t i l l , we do o c c a s i o n a l l y observe anomalous r e s u l t s and can only warn other workers t h a t d e a l i n g w i t h nanogram amounts o f i n d o l e s is d i f f i c u l t . A second method o f assay o f IAA has been developed by Mr. J . Cohen and i n v o l v e s a "double i n t e r n a l standard" u s u a l l y C - I A A and C - i n d o l e - 3 - b u t y r i c a c i d ( 3 7 ) . I w i l l not discuss t h i s method o f assay except to i n d i c a t e t h a t i t i s p o s s i b l e to develop assays not i n v o l v i n g mass spectrometry but w i t h comparable s e n s i t i v i t y and good s e l e c t i v i t y . k

4

4

U

11+

M e t a b o l i c "Turnover" o f P l a n t

Indoles

Ms. Pat H a l l and D r s . J . Nowacki and E . E p s t e i n have provided our knowledge o f the amounts and r a t e o f metabolic turnover of the i n d o l y l i c components o f the kernels o f Zea mays ( c f . 27, 28; Nowacki, unpublished; E p s t e i n , unpublished). This knowledge ïïâs enabled u s , 1) to i d e n t i f y the "seed auxin p r e c u r s o r " - - t h a t i s the compound which i s transported from the seed to the growing shoot ( 3 9 ) , and 2) has provided a p o r t i o n o f the proof t h a t IAAra/o-inositol and IAA are i n r e v e r s i b l e e q u i l i b r i u m i n the shoot tissue. Proving t h a t IAA and IAA e s t e r s are i n r e v e r s i b l e e q u i l i b r i u m i n the t i s s u e i s e s s e n t i a l i f we wish to p o s t u l a t e hormonal homeostasis. These experiments r e q u i r e d l a b e l e d IAA and tryptophan, which are a v a i l a b l e commercially, and C - l a b e l e d I A A - z m / o - i n o s i t o l . This compound was synthesized by D r . Nowacki by r e a c t i n g C-IAAimidazole w i t h m ^ - i n o s i t o l ( 4 0 ) . A p p l i c a t i o n o f these l a b e l e d compounds to corn k e r n e l s , followed immediately by homogenization of the t i s s u e i n acetone permitted us to determine the amounts o f each constiuent i n the kernel by the isotope d i l u t i o n method o f R i t t e n b e r g and Foster ( 4 1 0 · An extension o f t h i s method, whereby the kernels are incubated f o r v a r y i n g periods o f time a f t e r a p p l i c a t i o n o f the i s o t o p i c a l l y l a b e l e d compound permits determination o f the "turnover" o f the p o o l . Such data are shown i n 1I+

1 4


8


HYDROLYZED CORN

A-3717 389.2 27.7 % d

A

A-97II

^—

J I\

385.2 Av. 2 8 . 1

% d

4

4

A* 9 0 2 4

/Λ 330.2 Λ

|1

28.4%

/ ;\

1 7

4

Α · 31826

/!V

326.2 -

d

β

-— 9

TIME (MIN.)

Figure 2. Selected ion chromât ο gram of a mixture of the methyl esters of tetrafluorobutyryl IAA and d IAA. The IAA was from an extract of corn seedlings and the d -IAA added during homogenization. Retention time is in minutes and the masses monitored are 326.2 and 385.2 for IAA and 330.2 and 309.2 for d -IAA. The percent d -IAA has been computed by the area of the peaks at 330.2/326.2 + 330.2 (base peak) and 389.2/385.2 + 389.2 (molecular ion). r

h

k

h


1.

BANDURSKi

9


Table I I . They showed t h a t tryptophan, IAA and I A A - T m / s - i n o s i t o l are t u r n i n g o v e r - - t h a t i s made and then d e s t r o y e d , or used, at rates such t h a t t^ was 5, 3.2 and 12 h r s , r e s p e c t i v e l y . Such data permitted several important conclusions concerning the metabolism o f these compounds, f o r example, t h a t i t i s the IAA e s t e r s , and not tryptophan, which serve as a source o f IAA f o r the germinating seed and secondly t h a t the I A A - i n o s i t o i s are t u r n i n g over at such a r a p i d r a t e t h a t they must be i n e q u i l i brium w i t h the I A A - r a / 2 - i n o s i t o l g l y c o s i d e pool perhaps a c t i n g as g l y c o s y l a t i o n reagents ( E p s t e i n , u n p u b l i s h e d ) . TABLE II Concentration and M e t a b o l i c Turnover of Some I n d o l y l i c Compounds i n Zea Kernels

Compound

Incubation Time hrs

dpm/yg

0

31,000

4

8,900

8

5,400

0

15,200

8

5,000

0

935

8

590

IAA

Tryptophan

IAA-mz/o-inositol

The "Seed Auxin

Recovered Sp. A c t .

k hrs

- 1

hrs

0.22

3.2

0.14

5.0

0.06

12.0

Precursor"

Of great importance, f o r these s t u d i e s , was knowledge o f both pool s i z e and t u r n o v e r . T h i s knowledge permitted c a l c u l a t i o n of the s p e c i f i c a c t i v i t y o f the a p p l i e d i s o t o p i c a l l y l a b e l e d compound at any d e s i r e d t i m e . Thus, r a d i o a c t i v i t y , from a l a b e l e d compound a p p l i e d to the endosperm and appearing i n the shoot could be t r a n s l a t e d i n amounts o f compound moved from seed to shoot. For these experiments, minute amounts o f l a b e l e d IAA, t r y p t o phan, or I A A - r a / o - i n o s i t o l were a p p l i e d to an i n c i s i o n i n the semil i q u i d endosperm o f 4 day germinated Zea s e e d l i n g s . A f t e r 8 hrs o f i n c u b a t i o n , the shoots were harvested and the IAA, tryptophan or IAA-777z/0-inositol i s o l a t e d using r i g o r o u s p u r i f i c a t i o n t e c h niques. Now, knowing the s p e c i f i c a c t i v i t y o f the a p p l i e d compound at the m i d - p o i n t of the experiment we could c a l c u l a t e the


10

PLANT GROWTH

SUBSTANCES

average r a t e of t r a n s p o r t o f t h a t compound from endosperm to shoot. A summary o f a p o r t i o n o f these data ( c f . 27/, Nowacki, unpublished; E p s t e i n , unpublished) i s shown i n Table I I I . As can be seen IAA-Tm/s-inositol moves from endosperm to shoot at a r a t e of 6 p m o l s - s h o o f ^ h r . We had p r e v i o u s l y estimated t h a t about 9 p m o l S ' S h o o t - ^ h r " of IAA compound must be moving from endosperm to shoot to s u s t a i n i n d o l e concentrations i n the shoot (38) and G i l l e s p i e and Thimann (42)measured 5 p m o l s - s h o o t ^ - h r - of IAA d i f f u s i n g down from e x c i s e d Zea t i p s . On the assumption t h a t what goes down must come up, at l e a s t 5 p m o l s - s h o o f ^ h r " must be going up. By c o n t r a s t free IAA moves from endosperm to shoot at a r a t e of 0.015 p m o l - s h o o t " ^ h r - and tryptophan i n the endosperm appears as IAA i n the shoot at l e s s than 0.2 p m o l - s h o o t " · h r * . This l a s t f i g u r e i s high and c o u l d , i n f a c t , be zero s i n c e non-enzymatic conversion o f tryptophan to IAA occurs so r e a d i l y . Thus these data e s t a b l i s h t h a t IAA-mz/s-inositol i s the "seed auxin precursor" f o r Zea mays ( c f . 27_ and Nowacki, unpublished). - 1

1

1

1

1

1

1

TABLE I I I Rate o f Transport o f I n d o l y l i c Components From Endosperm to Shoot Compound a p p l i e d to endosperm

Compound i s o l a t e d from shoot

Rate pmols.shoot-^hr0.015

3

H-IAA

IAA + e s t e r IAA

3

H-tryptophan

IAA + e s t e r IAA

0.15

IAA + e s t e r IAA

6.2

lif

C-IAA-ra/0-inositol

1

The E q u i l i b r i u m Between IAA and IAA-myo-inosito! in vivo Dr. Hamilton e a r l i e r observed t h a t ether e x t r a c t i o n of t i s s u e induced a u t o l y s i s , l i b e r a t i n g a c t i v e esterases and g l y c o s i dases, and thus l e a d i n g to more free IAA than e x t r a c t i o n of t i s s u e by p o l a r , and t h u s , enzyme-denaturing s o l v e n t s ( 3 5 ) . Thus, we knew then t h a t there were enzymes i n the t i s s u e capabTe o f h y d r o l y z i n g IAA e s t e r s . Much l a t e r , Kopcewicz demonstrated the presence o f an enzyme system which could s y n t h e s i z e Ihh-myoi n o s i t o l from IAA, ATP, M g and CoASH (43). More r e c e n t l y , Mr. Lech Michalczuk (unpublished) has shown t h a t IAA-CoA w i l l a c y l a t e i n o s i t o l only i n the presence of other n u c l e o t i d e s . Thus, the r e a c t i o n i s complex, but there i s no doubt t h a t enzymes to make and hydrolyze the IAA e s t e r s are present i n c o r n . Now, are the enzymes a c t i v e in vivo! The data of Ms. Schulze and H a l l and Nowacki, E p s t e i n and Cohen (27_, 38; Nowacki, unpubl i s h e d ; E p s t e i n , unpublished) demonstrate t h a t they a r e . We + +


1.

BANDURSKi


11

showed i n 1972 t h a t there i s 93% e s t e r i f i e d IAA and 7% free IAA i n the shoots o f Zea s e e d l i n g s (26). The d e s i r e d experiment then was to apply l a b e l e d Ihh-myo-inositol o r l a b e l e d IAA to the endosperm of Zea k e r n e l s and determine whether we approach the same value--93% e s t e r , 7% f r e e - - s t a r t i n g e i t h e r from e s t e r o r free l a b e l e d IAA. H y d r o l y s i s or s y n t h e s i s o f e s t e r i n the endosperm becomes unimportant s i n c e the pools are so l a r g e t h a t we would not see a p p r e c i a b l e r a d i o a c t i v i t y i n the shoot i f h y d r o l y s i s or e s t e r i f i c a t i o n occurred i n the endosperm. Thus, we can determine whether e q u i l i b r i u m i s a t t a i n e d a f t e r the l a b e l e d compound enters the shoot. Mr. Nowacki a p p l i e d l a b e l e d I A A - r a ^ - i n o s i t o l to the endosperm and found 94% e s t e r and 6% free IAA i n the shoot (unp u b l i s h e d ) . Ms. H a l l a p p l i e d l a b e l e d IAA to the endosperm and found 70% e s t e r and 30% free IAA i n the shoot ( 3 8 ) . These values approximate those found f o r n a t u r a l in vivo c o n c e n t r a t i o n s by Ms. S c h u l z e . The conversion o f free IAA to e s t e r , as s t u d i e d by Ms. H a l l , are low but these were e a r l y experiments done before we were aware o f the ease o f h y d r o l y s i s o f the e s t e r s . The r e s u l t s demonstrate t h a t one approaches the same e q u i l i b r i u m amounts o f e s t e r and free IAA s t a r t i n g from e i t h e r compound. We use the word e q u i l i b r i u m to denote t h a t e s t e r IAA can be hydrolyzed to free IAA and free IAA can be converted t o e s t e r IAA. We do not imply t h a t t h i s i s a r e v e r s i b l e r e a c t i o n c a t a l y z e d by a s i n g l e enzyme ( 4 3 ) . This c o n s t i t u t e s the f i r s t demonstration i n b i o l o g y of an in vivo e q u i l i b r i u m between a hormone and i t s c o v a l e n t l y l i n k e d conjugates. Is the E q u i l i b r i u m Between IAA and i t s Conjugates P e r t u r a b l e by an Environmental Input An attempt to answer the question o f whether the environment c o n t r o l s p l a n t growth by p e r t u r b i n g the e q u i l i b r i u m between free and c o v a l e n t l y conjugated hormone i s the major e f f o r t o f our l a b o r a t o r y . To date only one environmental input has been t e s t e d and t h a t i s p h o t o i n h i b i t i o n o f growth. I t has been known f o r many years t h a t a b r i e f f l a s h o f l i g h t w i l l i n h i b i t the e x t e n s i o n growth o f an e t i o l a t e d s e e d l i n g p l a n t ( c f . 4 £ ) . The question then becomes, when p h o t o i n h i b i t i o n o f growth o c c u r s , w i l l there be a concomitant decrease of free IAA and a commensurate i n c r e a s e i n e s t e r IAA? The r e s u l t s o f t h i s experiment are shown i n Table IV. A 20 second l i g h t f l a s h r e s u l t e d i n a 43% i n h i b i t i o n o f growth as measured 90 minutes a f t e r the l i g h t f l a s h . The free IAA decreased by 35% and e s t e r IAA increased by a commensurate amount (45). Thus, our working hypothesis t h a t growth i s c o n t r o l l e d by the r e l a t i v e amounts o f free and conjugated hormone and t h a t i t i s t h i s r a t i o which r e f l e c t s the environment i s , i n t h i s case, confirmed.


12

PLANT GROWTH

SUBSTANCES

TABLE IV Photo-Induced Change i n Growth and i n Free and Free Plus E s t e r IAA Dark

Light

Δ

%

-1.1

-34

mm/90 mi η Growth

3.6

2.6 yg/kg

Free IAA

23

13

-10

-42

Free plus e s t e r IAA

68

77

+9

+11

An Hypothesis Concerning Hormonal Homeostasis The "take home l e s s o n " I wish to leave with you i s t h a t the hormones, IAA, the g i b b e r e l l i n s , c y t o k i n i n s , and a b s c i s i c a c i d , a l l occur i n free and conjugated form ( 4 £ , 47, 48, 49) and t h a t anything t h a t a f f e c t s the r e l a t i v e amounts of free and conjugated hormone w i l l c o n t r o l growth. A s i m i l a r h y p o t h e s i s , concerning mainly the g i b b e r e l l i n s has been made ( 5 0 ) . In the s p e c i a l case of IAA we have demonstrated t h a t IAA i s i n e q u i l i b r i u m w i t h i t s conjugates and t h a t t h i s e q u i l i b r i u m can be s h i f t e d by l i g h t . Thus, from these l i m i t e d d a t a , we propose, as a working hypothe s i s , t h a t the environment a f f e c t s the r a t e of p l a n t growth by causing changes i n the r e l a t i v e amounts o f free and conjugated hormone. This concept i s i l l u s t r a t e d d i a g r a m a t i c a l l y i n Figure 3. We envisage, Figure 3, t h a t environmental s t i m u l i , as f o r example l i g h t , heat, g r a v i t y , water s t r e s s , e t c . , impact upon one or more sensory apparatuses. In the case of l i g h t t h i s would be a pigment, whereas other s t i m u l i would impact upon the p l a n t counterpart of a " s o l i o n " ( 5 1 ) . A " s o l i o n " senses changes i n heat, sound, p r e s s u r e , g r a v i t y , e t c . , using a r e v e r s i b l e redox system and a minute a p p l i e d p o t e n t i a l . Since p l a n t c e l l s have a s u i t a b l e b i o - e l e c t r i c p o t e n t i a l (52) and redox systems i n t h e i r cytoplasm, they can be, i n a very r e a l sense, " s o l i o n s " . The sensor then t r a n s f e r s i t s s i g n a l , perhaps a hydride ion from a f l a v i n , to one o f the transducer enzymes. Chemically t h i s could mean using the hydride i o n to reduce a d i s u l f i d e bond i n an en zyme t h a t synthesizes or hydrolyzes hormone conjugates--thus changing the a c t i v i t y o f the enzyme. I f the h y d r o l y z i n g t r a n s ducer i s a c t i v a t e d then more a c t i v e hormone r e s u l t s . I f the s y n t h e s i z i n g transducer i s a c t i v a t e d then there i s l e s s free hormone and l e s s hormone e f f e c t o c c u r s . We do not know what hor mones do to c o n t r o l growth nor how many processes must occur f o r


BANDURSKi

lndole-3-Acetic Acid Conjugates

ENVIRONMENTAL

SENSOR

STIMULUS

TRANSDUCER HORMONE

CONJUGATE

HYDROLASE FREE HORMONE + X

[HORMONE-X] HORMONE

CONJUGATE

SYNTHETASE

I GROWTH SYSTEM

Figure 3.

Diagram of a system for control of plant growth by varying the relative amounts of free and conjugated hormone


14

PLANT GROWTH

SUBSTANCES

growth to r e s u l t so we show a dotted arrow l e a d i n g to growth. My personal f e e l i n g i s t h a t what the hormone does cannot be d i s covered u n t i l in vitro hormone-responsive systems are a v a i l a b l e (19). Here we encounter the Heisenberg u n c e r t a i n t y p r i n c i p l e as a p p l i e d to b i o l o g y . Conclusion I wish to c l o s e now upon an o p t i m i s t i c but cautious note. U l t r a - m i c r o s c a l e chemical assays o f l a b i l e p l a n t hormones w i l l never be easy and r o u t i n e . Nonetheless, use o f i n t e r n a l standards and the detectors now a v a i l a b l e f o r gc and h p l c , or the s e n s i t i v e and s e l e c t i v e mass spectrometer, permits assays of the hormones i n 1 to 10 gm amounts o f t i s s u e thus p e r m i t t i n g p h y s i o l o g i c a l experiments. We must someday return to b i o - a s s a y s , using a d e f i n i t i v e chemical assay and a p h y s i o l o g i c a l l y s e n s i t i v e b i o assay s i m u l t a n e o u s l y . This i s true because the t i s s u e s c o n t a i n both hormones and high amounts o f , u s u a l l y i n h i b i t o r y , phenylpropanes, as w i l l be discussed l a t e r i n t h i s symposium. A l s o , as Professor J . van Overbeek has i n d i c a t e d (personal communication), we w i l l not understand the physiology o f the organism u n t i l we simultaneously know what happens to a l l the hormones and t h e i r conjugates during complex developmental phenomenon. D i f f i c u l t as t h i s sounds, i t w i l l be p o s s i b l e , provided we confine ourselves to a few p l a n t s and s e r i o u s l y t r y to understand the hormonal system. L a s t l y , I b e l i e v e our working hypothesis o f hormones and t h e i r conjugates as homeostatic hormone systems w i l l lead to new and answerable q u e s t i o n s . I emphasize, however, our data apply to one hormone, and to one environmental input and only to seedl i n g Zea p l a n t s . W i l l there be a g e n e r a l i t y to our working hypothesis t h a t the environment c o n t r o l s the r a t i o of free to conjugated growth hormone and thus c o n t r o l s growth? We f e e l t h a t the answer to t h i s question has great i m p l i c a t i o n s f o r the cont r o l o f food and f i b e r production by a p p l i e d growth r e g u l a t o r s . I t h i n k we should work hard to answer t h i s q u e s t i o n . Abstract Most of the indole-3-acetic acid (IAA) in plants occurs as ester or amide-linked conjugates. This preponderance and apparent ubiquity led to a study of the functions of the conjugates. Evidence for four physiological roles has been found: 1) Conjugation i s reversible and provides the plant with a way to regulate its IAA l e v e l s , and thus i t s growth rate, in accordance with the environment; 2) one of the conjugates (IAA-myo-inositol) i s the chemical form in which IAA is transported from the seed of corn to the shoot, suggesting that conjugation provides the plant with information concerning where the hormone-precursor should be del i v e r e d ; 3) IAA conjugates serve as a source of IAA for the seed


1.

BANDURSKi


15

and seedling; and 4) conjugation of IAA protects it against peroxidative attack. To our knowledge, this i s the first case in biology where hormone levels are controlled by the formation and hydrolysis of a covalent bond. Acknowledgements The support o f the M e t a b o l i c B i o l o g y S e c t i o n o f the National Science Foundation PCM 76-12356 i s acknowledged. Without sust a i n e d support t h i s work could not have been accomplished. This i s j o u r n a l a r t i c l e 8963 from the Michigan A g r i c u l t u r a l Experiment Station. I am indebted to my colleagues D r s . Cohen, E p s t e i n , and Nowacki and to Mr. M i c h a l c z u k , Ms. S c h u l z e , and Ms. Hall f o r permission to use t h e i r unpublished d a t a ; to Ms. Joanne Di Lucca Schlub f o r valuable help i n manuscript p r e p a r a t i o n ; and to Dr. R. Chapman and a grant to Professor C. C. Sweeley (NIH-RR00480) f o r the use o f the MSU-NIH mass s p e c t r a l f a c i l i t y .

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Plant Growth Substances - American Chemical Society

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