.. ~ REPORT A

Professor:

THE AUSTRALIAN NATIONAL UNIVERSITY

RESEARCH SCHOOL OF BIOLOGICAL SCIENCES

DEVELOPMENTAL AND CELLULAR BIOLOGY

ANNUAL REPORT FOR 1968

STAFF

38/1969

D.J. Carr, B.Sc. Ph.D.(Manc.)., M.Sc. (Melb.) F.I.Biol.

Fellow:

J.D. Pickett-Heaps, B.A., Ph.D (Cantab). (From October, previously Research Fellow).

Honrary Fellow:

Stella G.M. Carr, B.Sc., M.Sc. (Melb.).

Research Fellows:

G.D. Clark-Walker, B.Sc.Hons, M.Sc. (Univ. of W.A.) .,

D.Phil (Oxon). (From December).

J.I.M. Stevenson, B.Sc. (Birm) ., Ph.D. (Edin). (From October)

Visiting Research Fellows:

L.C. Fowke, Ph.D. (Carleton, Ottawa). (From May)

T.P. O'Brien, Ph.D. (Monash). (From December)

Research Officer:

P.B. Goodwin, B.Sc.Agr. (Hons), M.Sc.Agr. (Univ. of Sydney),

Ph.D. (Nottingham). (From August)

Technical Officer:

Kathleen A. Britt, A.A.I.M.L.T.

RESEARCH WORK

Introduction

Developmental biology is concerned with all aspects of the

development of organisms, organs, cells and cell organelles.

Its methods range from the classical descriptive to the molecular.

A modern view would regard development as genetically programmed

and its study as an extension of the study of gene action.

However, a direct attack on problems of growth and differentia­

tion via studies of gene action is feasible only in exceptional

and possibly atypical cases.

There are, however, many levels at which attacks on problems

REPORT A -2- 38/1969

~ f development can be made, and some of these are exemplified

in the work of the group. Studies on organelle development are

widely favoured at the present time as promising an insight

into the integration of activities in the cell and the role of

extranuclear genetic information in development. The chloroplast

and the mitochondrion are the organelles of choice for such

studies since their development can be suppressed or induced at

will. The same ability to trigger a phase of development by

simple means - light or a hormone, for instance - makes higher

plants and insects organisms of choice in studies of even more

complex developmental mechanisms. The organization of cellular

ultrastructure by which these processes and others involved in

development are carried out is still poorly understood and often,

as in the work of the group on cell division, further progress

in understanding depends on the judicious choice of organisms

and the perfection of new techniques.

Cell Livision in Plants

Stomatal differentiation (Pickett-Heaps)

The fine structure of developing stomatal complexes was

examined in Commelina ana in whea t (Triticum} tissues that

had been subjecteu to centrifugation or treatment with caffeine.

The results all indicated that preprophase microtubule bands

are not functional in positioning the nucleus prior to division,

rather, they are thought to represent a response to the develop­

ment of polarisution in the tissue (induced in this case by

the guard mother cell). There is good evidence that these

microtubules arc incorporated direct into the mitotic spindle.

Cell Division in Oedogoniurn and Spirogyra (Pickett-Heaps and Fowke)

Developm~nt of new processing techniques has enabled a

fine-structural study of these two species to be undertaken.

Both show unusual or unique characteristics. Mitosis in

Oedogonium is preceded by flattening and elongation of the nucleus

(due probably to an enveloping sheath of microtubules) and

differentiation of the polar membrane. Complex kinetochores

appear at metnphase and anaphase separation is achieved within an

intact nuclear envelope. Telophase is characterised by considerable

REPORT A -3-

- . 'l 1 . 1· . . f s21na e e ongation ana E: imination o

38/1969

nucleolar material

as a 1·midboci.y". ThC;:n partitioning of thE: cE:11 is achieved by

a unique structure, the septum, composed of microtubules and

VE:sicles. 'l'hc cell wall at t.hE'. ring then splits circurnf eren­

tially, possibly cue to a build up of turgor pressure associated

with golgi activity, and the ring stretches to form the new

outer layer of the cell wall. The septum moves up the cell,

and finally the vesicles in it coalesce to form the new cross

wall.

In Spirogyra, preprophase is indicated by thickening of the

cyto~lasmic strands connecting the central nucleus to the peri­

pheral cytoplasm. At this stagei the strands fill with microtu­

bules, whilst wall microtubules decrease markedly in number.

At prophase, a highly oriented wall of cytoplasm is seen at

each polep and nucleolar dispersion is most characteristic.

At metaphase, chromosomes are detectable, disruption of the

nuclear membrane allows the nucleolar mass, containing the

chromosomes, to be invaded by the spindle microtubules.

During anaphase th<:: chromosomes are virtua.lly obscured by the

nucleolar material. The nuclei then reform. A septum is formed

during anaphase by annular furrowing of the outer membrane,

which evc.:ntually partitions the cell. however, ultrastructural

stuaies indicate that the last part of cell partitioning is

accompanied by a ~hragmoplast-likc structure on the inner edge

of th8 ingrowing septum.

Phytochrome-1.viediated P.hotomorphogenesis in Plants:

(Carr, Goodwini Menhenett, Crossley)

Red-light inauc~d loaf unrolling in barley {Carr, Goodwin,

Menhenett)

The first leaves of dark-grown gr3ss seedlings are tightly

rolled. They unroll after a brief perioct of illumination with

light, the most ~ff~ctive r~gion of the spectrum being the red

with a peak at about 660 run. Unrolling tal..es several hours.

anct results from differential cell growth. The effect of red

light is cancelled by bri~f irradiution with far red, with a

peak at about 730 run. E'e.r red reversal of a red response is

-4 - 38/1969

characteristic of a class of photomorphogenetic responses in

plants in which -c:.he absorbing pigment is phytochrome. Far

red reversal is possibl~ only within a limited period from the

ena of the effective red irradiation, i.e. irreversible processes

arc set in motion by the latter which lead on to unrolling.

In previous work it has been possible to show that one of these

processes is the synthesis of a gibberellin which can be extracted

and which IJroves to replace the need for rE:!ci light in inducing

unrolling. The gibberellin begins to be produced within 10 minutes

of tht start of rea irradiation and synthesis continues over

about 2 hours, the content of gibb~rellin declining after about

1 hour. Previous work had also shown that protein synthesis is

implicated both for the gibberellin synthesis and in its action.

Attempts are being made to set up this interesting system

(yreviously investigatea in Belfast) in Canberra, but difficulties

have been exverienced with unresponsive seed and impure light

sources. Some of th~ aifficultius may be due to modification

of the response by storage conditions of the seca, water regimes

in raising seedlings or climatic conditions of the physiological

darkroom. Recent work has shown that the physiological age of

the seeulings is an im~ortant factor in the response.

Attachment of roots to glass surfaces (Crossley).

E;arliE:r this year Ta nr!da had described an '· instant 1• phyto­

chrom~ response in which barley root tips gently swirled in a

complex medium in phosphate-coated flasks a ttached to the glass

after irradiation with reci light and detached after subsequent

irradiation witi1 far-red. The response was practically instant­

aneous and attributed to membrane changes resulting in electrostatic

attraction of the root tips to the glass. This system is typical

of some in which phytochrome-mediated responses are supposed to

be too rapid to permit of an explanation in terms other than

changes in membrane pt:!rmee.bility. Attempts are being made to

rept:at Tanada's work and, if they are successful, to analyse

the system.

Studies on Pa ramecium

-5- 38/1969

The role of the macronucleus in development (Stevenson)

Previous studies on Paramecium have been conc~rned with the

elucidation of the life history, mating and mating types,

flagellar antigens and symbiotic relationships. It has also

been shown that there are age-correlated changes during the life

cycle. The pur~ose of the studies just begun is to investigate

the control of these developmental changes. Despite the f~ct

that some of them are known to be regulated by single genes

the main control appears to rest with the highly-polyploid

macronucleus. The cuntrol by the macronucleus seems to depend

on sequential gene activation. Methods have already been developed

fer the isolation of macronuclei (Stevenson, Journal of Protozoology

14, 412-414, 1967).

Studies on Mitochondria

(Clark-Walker)

I•litochondria from all organisms studied to date have been

found tc contain DNA. Yeasts and Hucors are two groups of

organisms which particularly lend themselves to the study of the

function of mitochondrial DNA as they can both be grown fermen­

tatively under anaerobic conditions. In the absence of oxygen

bcth groups of organisms app8ar to loose functional mitochondria

which rt;;-appear . . . .. upon subsequent growth in air. This

characteristic is important fer answering the question c,f what

functional role the mitochondrial DNA has in mitochondrial

replication aRd the synthesis of mitochondrial components.

It is proposed tc study one aspect of the role of DNA

in mitochondria by using the antibiotic chloramphenicol. It has

been found that growth of yeust in the presence of this compound

results in the loss of cytochromes a-a3 , b & c 1 which are

typical mitochondrial compon~nts (Clark-Walker & Linnane (1967)

Journal of Cellula r Biology,~, 1 - 14.).

Further work is envisage.d as to whether chlo ramphenicol

leads to a suppressivn of mitcchondrial DNA replication and

whether this also occurs under anaerobic conditio ns.

REPORT A

- -6- 38/1969

Taxonomy and Biology of Eucalypts~

(Carr and Mrs Carr)

There are over 600 species and varieties of eucalypts

distributed over a large part of the continent and occupying

very many different ecological niches. They thus present excellent

material for the study of evolutionary taxonomy and of general

biology. The work, which has been in progress for some years,

is concern~d with both these aspects. This year, in addition

to the collection of additional material for the description of

a new Western Australian speci~s, the range of distribution

of a number of other species was notably extended. W.A. remains

the most interesting and at the same time least well-known area

of occurrence of eucalypts. Two main lines of work have been

followed in laboratory investigation this year~

Development of the flower in relation to classification.

The operculum of the eucalypt flower is a complex structure

which develops precociously and with m~ny specific variations,

which are of taxonomic value. Previous studies had revealed

two main patterns each associated with a group of species which

on other grounds also could be regarded as two separate genera

or sub-genera of the eucalypts. Rec~nt work has shown that the

simpler pattern, that ass0ciated with Eucalyptus s.s. is

6.evelopmentally complex. The more primitive members of this group

(hudesmieae) have now been shown tu have (Section A) an operculum

consisting only of the corolla or (Section B) an operculum which

is a composite structure derived from both sepals and p0tals,

fused so early and intimately that only in the earliest stages

or as a result of aberrations in development can thGir separate

contributions be aistinguished. The more advanced members

(Renantherae e.g.) have a pattern of development resembling that

of Eudesmieae Section B, but with development so telescoped

that the rese.mblance can be traced only in the earliest stages.

Anatomical characters of us~ in identification and taxonomy.

Since flowering material is not always available (or

accessible) anatomical characters of use in identification are

of potentially great value. An examination of certain features

"

I ~ -· REPORT A

-7- 38/1969

of the anatomy of 640 of the forms (species and varieties) has

been completed and the information is being prepared for publica­

tion. It mainly concerns the occurrence and distribution of

oil glands and ducts in various tissues of the stem and leaf.

Other studies involve leaf venation patterns which have

traditionally been used in a crude way as a means of identifica­

tion. The information content of the venation patterns is

clearly very great and only a fraction of it has hitherto been

used. The possibility of computer analysis of venation pattern

is being considered but first it is necessary to build up a

collection of cleared leaves of different species and to study

the way in which the venation pattern develops and varies

in selected species. Methods for clearing, staining and mounting

leaves to show the details of the venation pattern have been

developed. Other aspects of leaf anatomy are also being studied

and it is proposed to use for this purpose the newer methods of

resin enlbedding and ul trathin sectioning. These have already

given excellent results in preliminary work on the development

of oil glands.

Teaching and other activities

Two students are studying for a Ph.D. degree ; both supported

by A.N.U. Scholarships. They are Mr R. Menhenett from Reading,

lmgland, and t1r D. Crossley from York f England.

Publications

CARR, Stella G.H. 1 and CARR, D.J.

'Operculum development and the taxonomy of eucalypts 1

Nature , 219 1 513 - 515 (1968)

*·* + CARR , D.J. . 6 and REID, D .M . +

'The physiological significance of the synthesis of hormones

in roots and of their export to the shoot system '

in Biochemistry and Physiology of Plant Growth Regulators.

Ed. F'. Wightman and G. Setterfield. Ottawa (1968) p 215-236

** Based on work done prior to Joining this University

+ Not a member of this University

:. REPORT A - - 8- 38/1969

** GOODWIN, P.B.

'' Inhibitor of Receptacle Growth in non-pollinated

Strawberry fruit' . Nature , 218, 389-390 (1968)

PICKETT-HEAPS, J.D.

'Further Ultrastructural Observations on Polysaccharide

Localization in Plant Cells'. Journal of Cell Science,

~, 55-64 (1968)

PICKETT-HEAPS, J.D.

'Ultrastructure and Differentiation in Chara Sp 3. Formation

of the Antheridium ' . Australian Journal of Biological Science,

21, 255-274 (1968)

PICKhTT- HEAPS, J.D.

'Xylem Wall Deposition ~ Radioautographic Investigations using

Lignin Precursors'. Protoplasma f .§2_, 181-206 (1968)

PICKETT-HEAPS r J.D.

1 Ultrastructure and Differentiation in Chara Sp 4.

Spermatogenesis ' . Australian Journal of Biological Sci€nce,

~, 655- 690 (1968)

PICKETT-hEAPS, J . D.

'Microtubule-like organelles in the plastids or chloroplasts

of two algae'. Planta , 81 , 193 - 200 (1968)

+ ** + LAI"1B, A. J. +. , CLARK-WALKER , G.D . . , and LINNANE , A . W. +.

'The Biogenesis of Mitochondria 4 . The Differentiation of

Mitochondrial and Cytoplasmic Protein Synthesizing Systems

In Vitro by Antib iotics'. Biochimica e t Biophysica Acta ,

161, 415-427 {1968)

+ + ** REID, D.M. + ., CLEMENTS, J.B. +. u and CARR , D.J.

' Red light induction of gibberellin synthesis in leaves'

Nature , 217, 580-582 (1968)

Publications in the Pre ss

PICKETT-·HEAPS q J.D . and FOWKE, L.C .

'Preprophase microtubule bands in some Abnormal Mitotic

cells of Whea t 1• Journal of Cell Science.

+ Not a member of this University

** Based on work done prior to joining this University

:

. EPORT A 38/1969

-9-

PICKE'l'T- HEAPS f J . D. and FOWKE , L.C.

i Preprophase microtubules and stomatal differentiation

in Commelina'. Australian Journal of Biological Science .

PICKETT- HEAPS , J.D. and FOWKE r L . C.

1 Prepropha s e microtubules and stomatal differentiation ;

some effe cts of centrifugation on asymme trical cell division '.

Journal of Ultrastructure Research.

PICKETT- HEAPS, J.D. and FOWKE, L.C.

' Cell division in Oedogonium.I '. Australian Journal of

Biological Science .

+ + ** LASCELLES, J +. 1 RITTENBERG, B +. , and CLARK- WALKER, G.D.

'Growth and Cytochrome Synthesis in a Hemin-Requiring Muta nt

of Spirillum Itersonii' . Journal of Bacteriology.

** STEVENSON , J.I.M.

'The Biochemical status of mµ p a rticles'. Journal of general

Microbiology.

+ Not a member of this University

** Based on work done prior to joining this University