Auxin action and cell elongation : a rational approach : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Physiology at Massey University

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AUXIN ACTION AND A RATIONAL

CELL ELONGA'riON:

APPROACH.

A the sis pre sented in part ial

fulf illment of the re quire me nt s for the degree

of Doc to r of Philo s ophy in Plant Phys i olo gy a t

Massey Univ e r s. i ty.

Pauline Eli zab e th Pe,nny 1 9 70

To the c ourte ous and we ll wi lling read e r'S :

Al tho

�

^{h my} p a ine s hav not beene spent

(

c ourte ous Reade r

)

ⁱⁿ t he gra c i ous d i sc ove r ie of go lde:n Mine s , nor in the trac ing af t e r s i lver voine s , whereby my na t ive country might be inriche d w ith such merc han­

d i s e a s i t ha th mo s t in re que st and admira t i on; ye t ha t h _my lab our ( I t ru s t

)

been o t he .rwi se prof itab ly imp lo ied, in de s c rying of such a harme le ss.e treasure of herb e s, t ree s , and p lant s, a s the earth f ranke ly w i t hout violenc e off e re th unto o ur mo s t nec e s sa ry use s . Where in though myne a r t b e not ab le t.o

c onterva ile Na ture in her l iv e ly p ortra iture s, ye t

have I

c ounterf e i ted likene ss f or life , shapes and shad ows for sub stance o Ye,t may my b lunt at tamp t se rve a s a whets.tone t o se.t a n edge upon sharpe.y wits, by vihom I wi sh this c o ur s e D i s c ourse . might b e b oth fined a nd ref ined. Fault s,�� I c onfe sse have

e sc apQ.dSl some thro ugh d efe:et s in _my se.lfe t o perf orm s.o gre a t a work, and s o me by means of the grea tne ss, of the La·bour, b eing v o id of f r iend s. to beare s o me part of t he b urden. I trus t tha t the b e .st and we:ll minded will no t rashly c ond e mne me . Theref ore. ac c ep t t h i s a t my hand s

(

lov i ng c ountryme n

)

as a t o ke n of my goodwillo

John Ge.rard , 1 59 7

TABLE ^{OF CONTENTS}

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ACICNOvVLEDGEMENTS ^{• • • • •• • . • o o • o • o • o o • o • • o • o • • •• • o • • o • •}

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TABLE OF ABBREVIATIONS ^{• •• • • . o • • • • • • • • o • • • •o • • • • • • • • •}

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IliTRODUCTION ^{o • o o o • a o} • o o � o o o o o • o o o • o o o o o c o o o o C; o o o • • • • 1

PART I ^The Kine ti c s of Auxin Induced Cell _Elongation

A. Ih trod uc ti ono ^{o a e e • o • o • • " e • • o o • o • •o o o • • • • o • • •} 8 B ^• Ma tar ia ₁ s and Method s

1 ^• !'flat er i a 1 s ^{o • o o • • •• • o •• • • • o • • o • o • o • o • • • o • • •} 1 3 2 ^• :rvie thod s. ^{• •} 0 0 0 e 0 0 • 0 0 •• 0 0 0 • 0 0 • 0 0 0 0 • 0 • • • • • • • 1 3

a . For me a suri ng the growth of inta c t plant s 1 4 b. For b ioassays ^{• • • • • • • • • • • • •• •• •• • • • • • • • • •} 1 4 c . For l ong term kine t ic s ^{• • • • • •• o . .. . . . . . . .} 1 5 d. For short term kine ti c s ^{• • • • •. • •• • • • • • • • •} 1 5

1 ^• Long t e rm kine G

ic

^s

a . Growth of Inta.c t Plant s ^{• • • • ••• ••} o . . . 22:

b . Long term kine;tics of exc i sed segment s. . 2·4 2 . Short Term kine ti c s

a. Effec t of IAA o n the growth ra te of lupin hypoc otyl s�gments.

i. IAA pre se,nt cont i nuously ^{• •• • •• • o . . . . .} 26

ii. Short term

IAA

^{treatments, •• o • • • • • • • • • 26} i i i . Eff e c t _of l ight on I.A.A.-induced growth

rate ^.... ^.. ^{. o • o • o • • • o o o • • o o • o • o • • • o • • •} 29 iv. Eff'e c t o1� phospha te bufl'er. . . . ... . 32

D.

Page;

v. Experiments without a buffer solution. 3,2 b. Effect of other auxins on the growth rate

of lupin hypocotyl segments ^{• •• •• • . • • • • • •} 35

i. 2..;4-dichlorophonoxyacetic _acid ^{•• • • • • • o} 35 ii. !ndoloacet:::tmid� ^{•• • • • • • o • • • • •• • •• • o • • • •} 36 iii. Indolebutyric acid ^{•• • • • • o • • o • •• • •• • • •} o 36

iv. illdoleace.taldehyde 9 naphthalacotamide.

naphthoxyaco.tic acid, ethyl-indol-3-

yl-acetata ^{•• •• •• o • •• • • • • • • • o • o • · · · · · · ·} 39 c. Effect of au xins on the growth of pea

stern segments.

i. IAA o o o o o c o o o o e ^{• • o e o o o • o o o •} o o o o o o o o o • o o 39 ii. 2..94-dichlorophenoxyacetic acid _and

indoleacetarnide ^{• •• •• • o • • • • •• • o • • • • • • • • •} 41

, ,

Re s ume ^{• • o • o • o • • • • o o o • D • • • • o • • • o • • • • • • • • • • • • •} 4 1 PART I I The effect of auxin on macromolecules.

A. In trod uc t i on. ^{. . . . . .. . . . o •• •• • • • • •• • • • o} .. ^{o • o • •} 4 9 B. RNA synthesis

1 . Effect of actinomycin D

a. Long term kinetics ^{•• • • • • •• • • • • . • • •• • • • • •} 5 9

b. Short term kinetics ^{• • • • o• • • •• •• • • • • • •• • •} 6 1

2. �Yfect of IAA on RNA synthesis

a. Experimental design ^{• • •• • • •• • • • •• • • • • •• ••} 62

b. The short term effect of _IAA on _RNA

synthesis: ^{. .. . . • . o} . ^{. o} ........ a . . . 64

c. The small radioactivity peaks in the

r-RNA region of the ge.ls. ^{o . .. . . . . . . . . . ..} 67

Page:

d . Up take of' 32 P •••• ^{e • •} • • • • • • • ^• • • • • • • • ^• • • • • • • 70

C . Protein S:ymthe,sis

1 . Tho ef'f'ect of' c yc lohex imide on IA�- induced growth •••••.• ⁰ • ⁰ •••• ^{0 0} • ^e •• ⁰ •• ^{0 0} ••••• ⁰ • ^{0 e} • • 7 3 2. The ef'f'cct of' IAA on prote in synthe s is

a . Experi menta l d e s ign •••••••••••••••••.••• 77 b. Re sult s .. . . . , . . . e ••••••••••••••••• ^e • • • 7 8 3. Ef'f'cc t of' cyc loheximide on respirati on •••• 8 3 4. The ti ming of' the eff e c t of cyc lohe xi mide . 8 7

a. Pretreatment in IAA . . . .. . . .... . . 87 b. Pre treatment in cyc loheximide ••••••••••• 88 i . Me thod •••••••••• ^o ••• ^{o o} • ^o • • ^• • • • • • • • • ^• • • 9 0

i i. Re sults •••••••••••eoooooooooooooooooo 90 DISCUS S.I O N ••••••• ^o • ^c ••••••••••• ^o • ^o ••••••• ^o • ^• • • ^• • • • • • • 9 5

A. Short term kine t ics

1 . Comment s on me thod s . . .. . . 95 2 . The growth rate curve ••••••••••••••••••••• 1 02

3 . Exp lana t ions of' the growth rate c urve ••••• 1 07 4 . "Endogenous growth" . • • • • • • • • • • • • • • • ^{• •} • • • • • 1 1 1 B . B ioche mical Theorie s

1 . Category I - RNA synthe s is. i s re quired •••• 1 1 4

2 . Ca t e gory II- Pro t e i n synthe si s i&

re quirGdooooo••••o••••••••••••••••o••••••• 1 1 8

c. The primary s i te of' auxin a c t ion ••••••••••••• 1 2 3 D. A model for the primary mechanism of' auxin

a c t ion. ⁰ • ^{0 . e ' '}'',''' ••• ⁰ •••••••••• ⁰ • ⁰ ••• 0 . . . . 1 27 APPE1�IX I - Add itiona l examples of' kine tic

experiment s ••••••••••••••••••••••••••• ,.. 1 39

Page, .APPENDIX II - Mater-ials and me thods for Part I I . ... .. . 1 48 A. Incub a tion of plant t is s.ue ^{• •e ••• 0 e • 0 • 0 • 0 0} 0 0 0 0 1 48

B. Extrac tion of me.c romolecules

1 ^o Unde na tured Rl\fl1. ^{• •• • ••• • ••• o •• ••• • •• • •• o • • •} 1 48 2 . Total RNA ^{• • o e • ••• o o • • • ••• •• o • • o •••• •• o o •••} 1 5 0 3 . Orga nic phos.pha tes,·�··· ^{• •• • • • ••• •} 1 5 1 4 . Pro tein ^{•• • • ••• • •• ••• • •••• • • ••• • • •• • •• • ••• •} 1 5 2

c. Elec trophoresi s..

1 . Polyac rylamide ge l e le c t rophoresis ^{••• ••• ••} 1 5 3 a ^• RNA o o o o o • o • • • •o •• o • • • • • • • • ., • • o • � o • • a • • • • o 1 54 b . Prote in ^{• • •• •• ••••• • •• •• •• •• • • • ••• •• • • • •••} 1 54

2:. CelJ.ulose acc.tate ^{.•• • • • •• • • •• ••• •• • • •• •• •••} 1 54 D. Column c hroma.tography' ^{••••• •••• ••••• •• ••••••••}

1 . Me thyla ted a.lbumin Kie. selguhr

(MAK)

^{. . .. .. . . 1 .5 5} 2 . Hydroxyapa t i te ^{• • ••• • • • • • . • •• • •• •• • • • • •} ' ^{• • •} 1 5 5·

E . Determinat ion of RNA

1 ^• Tot a l RNA in extrac t s ^{• • •• • • •• • .• ••} 1 56 2 . RNA in ac rylamidc gels ^{• • • •• •• ••••• • • • •••••} 1 56 F. Deter-mina t i on of protein in acrylamide ge ls, ^•• 1 5 7

G. Date rmina t ion of rad ioac t iv i ty ^{•• • • •• •• • • • ••• •} 1 58 H . Measurement of resp ira t i on ^{• • • • ••• •• • ••• • ••• ••} 1 58

I . Prepara t ion of t issue. for electron

microscopy . . . . . . . . .. . .. . . .. . . . . .. . . . . .. . . . . . .. 1 ;5 9 APPEND LX I I I

APPENDIX IV

Detection of RNA on ac ry]amide ge.ls,. . . 1 60

The ft'ast running non-RNA p.eaks . . . . ^{• • • •} 1 63 BIBLIOQ-RJ:ll'JfY= ^{••• o • e. • •• o •• •.•••• •} • . ^• • • • • •. • • • • • • • • • • • • • • • 1 6 8

ABST RACT

A me,thod was developed to measure every: minute the growth of' a single segment excised from the elongating region of' a plant. The method was used to determine the short term kinetics, of growth in response to auxin addition. The method is not dependant on the use of' hollow cole.optile tissue and the results are plotted as a growth rate agains.t time. The technique has a

resolution _qn order of' magnitude higher than those in current use.

The results show that there i s a latent phase

before auxin-induced increase in elongation rate occurs.

After this latent phase9 there is a rnp.id rise. in rate to a maximlli� followed by a decrease and then usually a rise to a s,econd maximum. Three,

hyJJ

othe.ses for

explai�ng the growth rate curve are considered.

rt was found that neither RNA nor protein synthesis we·re required for the initial action of' auxin but that protein synthesis became necessary within a few minutes after auxin addition. The apparent half'-lif'e of' the protein whose synthesis is stimulated by auxin is about

1 2 min. This short half' _life. suggests that, after the synthesis of' the protein, there is a limited time during which it can act with auxin to increase elongation. A

mode.l which incorporated these results. has bee,n proposed.

and its relationship to the three hypotheses for exp1aining the growth rate curve is discussed.

( 1.)

A C K N 0 W L E D G E M E N T ₈

I wish to re c o rd and acknowled ge with thanks the

�ont inua l e nc ourage me nt of Pro f. R. G . Thoma s for research i n h is depar tment in gene ral and his interest in this proje c t in par tic ular .

Some of the research in this thesis. wa s. carried out in the lab ora t ory of Ji.vi'. J.\'1. Lyt t le to n a t App l ied

Bioche mi stry Divisi on, D . S . I.R. It was Dr . Lyt tle ,ton's ge ne rous s.haring of h is lab orat ory apace and e quipment which made so me asp e c ts of this wo rk fe as

*-

^{ble . During}

my f' ive month so jurn a t D . S.I.R. 9 I had many s t i mula t ing d iscussions with many of the profess·ional staff. For

this I vm·uld like to thank the m and I wo uld 1ike to t ha nk in part icular Prof. J . K . Heyes who also taught me the techniques of RNA extra c t i on and ac rylamid e ge l e le c trophores is; Dr . M . Clarke who al s.o taught me the

uae of _MAK c olumn c hroma t ography and c e l lul o se ac e ta t e

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electrophoresis'; and Dr. W.S. Sutton who also taught me the use of' hydroxyapatite' ch.rorna tography.

The ingenuity and helpfulness of' Mr.C. Baggers _and Mr. M. Mannering in the de-sign and cons,truc tion of' the

chamber are gratefully acknowledged. I also wish to thank Dr. J.P. Kerr and Dr. H. MacPherson, of' Plant Physiology Division, D. _s. I. R. f'or the light intensity

measurements.

I would like to thank. Mrs. K .F. Miller and Mr. D.

C. Marshall f'or doing some of' the later kine.tic experi­

ments and Mr. C.N. Wyatt f' or technical as.sistance.

Ideas are very seldom generated in vacuo _and perhaps the best way for generating them is in conversation with others. I wish to thank the many people who have

contributed in such an irilil'ect manner. In particular, the many "discuas:ions" with Vasil Saraf is, who see:S problems f' rorn a botanist 1 a point of viev1, hav.e helped clarif'y ideas and suggeated new lines of' appro ach; to my husband, David, who sees the fores,t in spite of' the tree,s; and to Kathy Miller who is very astute at finding errors in reas.oning and typescript.

To .._\n.n and Kim I can only say thamk-you - you will understand

in

a f' ew years ^.•

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D NA D OC IAA NA RNA

m-R...l�A r-RNA s-RNA TCA 294-D

TABLE OF ABBREVIATIONS

deoxyribonucleic acid deoxycholate

in

p

dol-3-yl- acetic acid numerical aperture

ribonucleic a·cid me s se nge r Rl'l"A

riboaomal _RNA soluble RNA

trichloroacetic acid

2 ,4- d ichlorophenoxyace t ic acid

( iv)