14
Plant development
Synthesis of chalcone synthase as the rate-limiting step in
phytochrome-mediated anthocyanin synthesis in mustard cotyledons
R. Brodenfeldt and H. Mohr, Biological Institute II, University of Freiburg, D-78 Freiburg, West Germany
Light- (phytochrome-) mediated induction of anthocyanin (aglycone cyanidin) may be considered a
biochemical model process of photomorphogenesis in seedlings of higher plants. While evidence was
obtained that physiologically active phytochrome (Pfr) stimulates synthesis of enzymes related to
anthocyanin biosynthesis, the identification of rate-limiting enzymes has not been successful so far. As an
example, the extractable amounts of key enzymes of flavonoid biogenesis such as phenylalanine ammonia
lyase (PAL) and chalcone synthase (CHS) in mustard (Sinapis alba L.) cotyledons do not correlate with the
rate of anthocyanin synthesis. - In the present work it was shown first that the assayable level of PAL and
CHS correlates with the amount (pool) of enzyme as determined by immunotitration. Secondly, the
turnover rates of the enzyme pools were determined after turning off the light signal by reverting Pfr back to
the physiologically inactive Pr with a saturating long wavelength far-red light pulse. It was found with both
enzymes that the turnover rate remains constant over the whole experimental period (PAL: t$ = 13,3 h;
CHS: tj = 11,1 h). - Thirdly, the rate of enzyme synthesis (ks) was calculated from the time course of the
enzyme level and from the turnover rate. It was found that the time course of kg correlates with the time
course of the rate of anthocyanin accumulation in the case of CHS but not in the case of PAL.
Correspondingly, an integration of ks (CHS) over time yields a curve which represents the time course of
anthocyanin accumulation. If the Pfr signal is turned off, the resulting changes in the rate of anthocyanin
accumulation reflect the changes in ks (CHS). It is concluded that the rate of anthocyanin synthesis is
determined by the newly synthesized CHS, while the rate of anthocyanin synthesis is not related to the total
pool of the enzyme.
Somatic hybridisation studies with a nitrate reductase deficient mutant
line olNicotiana tabacum L.cv. Gatersleben
S. Cooper-Bland 1, A. Kumar 2 and E. C. Cocking 1.1Plant Genetic Manipulation Group, University of
Nottingham, Nottingham NG72RD. 2Department of Biochemistry, University of Southampton,
Southampton S093TU.
Somatic hybridisation by induced fusion of protoplasts, with subsequent selection of viable heterokaryons
capable of plant regeneration, provides a new means of overcoming many incompatibility barriers and can
allow the production of novel plant genotypes. A major problem in somatic hybridisation is the availability
of a suitable selection system. In this present study we have used a selection system based on the use of
auxotrophic mutant lines of N. tabacum provided by Miiller. These nitrate reductase deficient mutants of N.
tabacum are of two types, viz those with a defective structural gene for nitrate reductase apoprotein (coded
nia mutants) and those with a defective synthesis of molybdenum cofactor (coded cnx mutants). The
mutants have been characterised by showing complementation to an active nitrate reductase following
protoplast fusion between the different nia and cnx mutants using a high pH calcium fusogen. Further
investigations have been carried out to demonstrate the amount of genome which can be transferred from an
irradiated donor protoplast (N. glutinosa) to a non-irradiated recipient protoplast (N. tabacum mutant nia
130) via protoplast fusion. Suspension protoplasts of N. glutinosa were given a 50kR y-irradiation dose (to
stop cell division) and were fused with mesophyll protoplasts of the regenerable nia 130 line. Hybrid
colonies were selected as nitrate utilising events and plant regeneration attempted where possible. A
comparative experiment fusing non-irradiated N. glutinosa protoplasts with nia 130 was performed and
plant regeneration of nitrate utilising events obtained. These two types of hybrid plants have been
characterised at the morphological (leaf and flower structure), cytological (chromosome number) and
biochemical (Fraction 1 protein, isozymes) levels and are shown to be hybrid. Results and a full discussion
of the effects of irradiating a protoplast fusion partner on hybrid genotype is presented.
Plant development
15
The use of macromolecular markers to study embryo development and
germination in Brassica nap us L.
Martha L. Crouch*, Anne E. Simon and Ruth Finkelstein, Department of Biology, Indiana University,
Bloomington, Indiana 47405, USA
We are studying the regulation of embryo development in Brassica napus L. (rapeseed) by comparing the
behaviour of embryos cultured under various conditions to their normal development in the seed. We
monitor the progress of embryogeny by the accumulation of storage proteins, using antibodies to measure
storage protein synthesis and cDNA clones to determine storage protein mRNA levels. During normal
development of embryos in seeds, storage proteins begin to accumulate in cotyledon and axis cells during
the last half of embryo growth, and they are broken down rapidly upon germination. Neither storage
proteins nor their mRNAs are present in non-embryonic tissues. Cultured embryos require either 10~6 M
abscisic acid (ABA) or high osmotic activity in the medium in order to continue high rates of storage protein
synthesis. Without these factors the embryos germinate precociously, but young embryos do not develop
into normal seedlings. Various abnormalities occur, such as the production of extra cotyledons at the shoot
apex, secondary embryos on the hypocotyl and cotyledons, and continued synthesis of storage proteins in
leaves and roots. Therefore, it appears that embryonic and germination processes are occuring concurrently
during precocious germination.
We have established a permanently embryogenic culture from the secondary embryos that form on
precociously germinating zygotic embryos. In this culture, new embryos arise from surface cells of old
embryos. The cultures have maintained embryo formation for at least 14 subcultures (one year). These
cultures synthesize storage proteins continually, which indicates that during normal embryo development a
signal is required to turn off storage protein synthesis as well as to turn it on.
The influence of growth regulators on the culture of protoplasts isolated
from habituated cotton callus
O. M. El-Shihy and P. K. Evans, Biology Dept., Building 44, Southampton University,
Southampton SO95NH
Protoplasts can be enzymatically isolated from habituated callus of Gossypium barbadense cv Giza 75.
After 3 days in culture medium lacking growth regulators some 52 % of protoplasts regenerated a cell wall
and first divisions were apparent after 7 days in culture. Approximately 9 % of the protoplasts embarked
upon division. Cell wall regeneration and cell division were influenced by a variety of factors, including light
intensity, temperature, culture density and medium composition. In the presence of low levels
(0-1-0-5 mg/1) of auxin either as IAA, NAA or 2,4-D or cytokinin either as BAP, Kinetin or 2iP cell division
was enhanced but not cell wall regeneration. Levels of 1-0 mg/1 or over, reduced both cell wall regeneration
and cell division. Higher concentration such as 5-0 mg/1 completely inhibited cell division whereas, a
concentration of 10 mg/1 (and in some cases less) completely inhibited both cell wall regeneration and cell
division. The most suitable growth regulator levels for cell division were Kinetin at 0-1-0-5 mg/1 which
caused 15 % of the protoplasts to divide or BAP at 0-5 mg/1 or NAA at 0-1 mg/1 or IAA at 0-5 mgyj causing
12-13 % to divide. In contrast, the addition of 2iP and 2,4-D resulted in reduced levels of division, and
completely inhibited cell wall regeneration and cell division at a concentration of 2-0 mg/1. The addition of
low concentration of growth regulators did shortened the time needed for cell wall formation and the time
between protoplast isolation and the onset of cell division. At low levels of growth regulators, 0-5-1-0 % of
the protoplasts embarked upon division and formed colonies of 6-10 cells after 3 weeks, but no further
growth was observed. After 'dilution' with fresh medium of the same composition, the percentage of colony
formation increased but no callus was obtained. However, on 'dilution' with fresh medium lacking either
growth regulators or mannitol, colony formation was enhanced and callus was obtained after 6-8 weeks.
16
Plant development
Growth and development of habituated cotton callus: delay in
senescence and stimulation of growth at a low temperature caused by
exogenous growth regulators
O. M. El-Shihy and P. K. Evans, Biology Dept., Building 44, Southampton University,
Southampton SO9 5NH
Callus can be produced on excised petioles of Gossypium barbadense cv Giza 75 when they are cultured
on M/S medium with 2 mg/1 IAA and 1 mg/1 Kinetin at 30 °C in light (3000 Lux). Whilst callus was
developing on excised petioles, the cultures were heat shocked for 2 days at 40 °C. After returning the callus
to the normal incubation temperature of 30 °C, some callus developed which grew rapidly and differed in
colour and texture to that normally produced. This callus was transferred to a medium lacking growth
regulators whereupon it continued to proliferate. Relatively large pieces (lg.) of this habituated callus were
subcultured every 2 weeks. At first, growth was rapid, but declined after 2 weeks, the tissue becoming
senescent. When growth regulators were included in the medium, this senescence was delayed, some
cultures continuing to grow for up to 4 months. However, in the presence of cytokinin, either as BA,
Kinetin or 2iP growth over the first 2 weeks was reduced. Reduction in growth, although to a lesser extent,
was also recorded when auxin, either as IAA, NAA or 2,4-D was present, but the addition of both auxin
and cytokinin reduced growth to a lesser extent than when included individually. However, in some
treatments the final yield of fresh weight was more when growth regulators were present than in their
absence. The delay in the onset of senescence as the result of the presence of growth regulators was related
to their concentrations, higher concentrations caused longer delays. At a temperature of 22 °C or less, no
growth of the habituated callus was observed, whereas, in the presence of some growth regulators (IAA or
Kinetin or a combination of these growth regulators), growth took place at the lower temperature of
20-22 °C.
In vitro continuous plantlet regeneration via somatic embryogenesis in
Hyoscyamus alb us L.
Maria Ludovina, L. Guimardes and J'. Montezuma-de-Carvalho, Botanical Institute, University of Coimbra,
3049 Coimbra, Portugal
During experiments in anther culture of Hyoscyamus albus in different media an aberrant line of plantlets
was isolated from one anther that had been pre-treated in a medium containing caffeine. We describe here
the preliminary results about the behaviour of such plantlets. It was observed that these plantlets in a
hormone-free basal medium have the capacity of regenerating new plantlets via somatic embryogenesis. The
embryoids, whose somatic embryo character was supported by histological examination originated primarily
on the margin of young leaves. This production of embryoids is observed both in solid and liquid media
maintained at light or in darkness. The frequency of such morphogenesis is not dependent upon the number
and the length of previous cultures passages. In order to see the effect of some plant growth regulators on
such morphogenetic
- . _ _ . _ _ .
-.
far the data indicate
and transform
genesis; 1BAP plus 2,4-D stimulated
embryogenesis at 0-0001 to 0-1 mg 1 ) but depressed it above
0 1 mg r and at 10 mg I"1 an amorphous callus is obtained; GA3 (0-05 to 10 mg I"1) stimulated
embryogenesis; Amo 1618 (at 0*05 to 10 mg I"1) greatly reduces the number of regenerated plantlets. Work
is underway to determine chromosome number of the regenerated plantlets.
Plant development
17
Pollen embryogenesis: a model system for the life cycle of higher plants
Erwin Heberle-Bors*, Department of Applied Microbiology, State University of Agriculture and Forestry,
Peter-Jordanstr. 82, A-1190 Vienna, Austria
In vitro cultured pollen of tobacco are able to produce embryos which develop into haploid plants. Only a
particular fraction of the pollen is competent, and competence is acquired before culture in vitro, in the
anthers of the pollen donor plants.
For the donor plants, the embryogenic pollen grains are functionally sterile pollen grains, and they are
produced in high frequency in flowers in which sex balance is shifted towards femaleness. High number of
embryogenic pollen grains and feminization occur under environmental conditions which reinforce floral
induction. Both are, however, not induced during floral induction period, but during post-induction flower
development, before meiosis. Thus, during microsporogenesis, two alternative developmental pathways are
open: either gametophyte (normal pollen with pollen tube and sperm formation) or sporophyte formation
(embryogenesis). Direct formation of a sporophyte from pollen can be interpreted as maximum reduction of
the male gametophytic phase in the alternation of generations in higher plants, in analogy to embryogenesis
from the female gametophyte, the embryosac (zygotic, parthenogenic, apogamic). In fact, pollen
embryogenesis is nothing else than apogamy (sporophyte formation via embryogenesis from somatic cells of
the gametophye). Since pollen is produced in much higher quantity and since there is much easier
experimental access to pollen than to embryosacs, pollen embryogenesis seems to be valuable experimental
system for studies on 'oogenesis' and embryogenesis in plants. In addition, pollen embryogenesis has an
applied aspect, since haploid plants are of great importance for plant breeding.
With the presented model, it is possible to see the early processes of sexual reproduction in plants
analogous and converging to the respective processes in animals, without neglecting the peculiarity of an
alternation of generations in plants.
Interactive systems and sequential cell differentiation in angiosperm
reproduction
/. Heslop-Harrison*, University College of Wales, Welsh Plant Breeding Station, Plas Gogerddan,
Aberystwyth, DyfedSY233EB
Several of the tissues involved in reproductive development in angiosperms show sequential differentiation, the cells passing often quite rapidly from one function to another, usually according to a strict
time-table. This is most clearly seen in secretory tissues of the anther, ovary, stigma and style, where
different products are often seen to be synthesised and released in successive waves. The behaviour is often
closely correlated in immediately contiguous cells even when these are pursuing different programmes, and
this raises the question of how the co-ordinated timing is maintained. Two possible interpretations will be
discussed, (a) that the chronology of events is established in each tissue by endogenous timing devices,
activated and perhaps occasionally re-synchronised by non-specific signals, and (b) that correlated,
stepwise, progressions in contiguous cells pursuing different pathways of differentiation depend upon a
continuous exchange of 'information' about the status of each.
18
Plant development
The biology and biochemistry of lectin-mediated recognition in the
Rhizobium-legume association
/. W. Kijne*, Department of Plant Molecular Biology, Botanical Laboratory, University of Leiden,
Nonnensteeg 3, 2311 VJ Leiden, The Netherlands
Successful infection of leguminous plants by the gram-negative bacterium Rhizobium under low-nitrogen
conditions in the soil leads to the development of nitrogen-fixing root nodules. The Rhizobium-legume
association shows various degrees of host-specificity (e.g., clover roots are susceptible to R. trifolii, but
resistant to other rhizobia). The genes involved in rhizobial infection and thus in host-specificity occur on
large, so-called sym-plasmids.
Several observations indicate that attachment of Rhizobium onto the top of growing root hairs is the first
specificity-determining step in the infection process. The molecular basis of host-specific attachment is
suggested to a specific binding of certain sugar sequences on the rhizobial surface by lectins on the root hair
top (for recent reviews, see 1 and 2). Especially rhizobial capsular polysaccharides and LPS have been
mentioned to contain lectin receptors. The current state of the lectin-recognition theory will be summarized.
Infective rhizobia excrete biologically active molecules which affect root hair development (e.g., 3). The
action of these substances can be defined as induction of tip growth (Van Batenburg et al., in preparation)
and leads to a variety of root hair deformations depending on the position of the rhizobia relative to the root
hair. The active molecules might be considered as growth signals. At present it is unclear whether one or
several substances are involved. One of such growth signals, a clover root hair branching factor, has been
characterized as a class of oligosaccharides (4). Interestingly, the induction of root hair deformations shows
roughly a similar host-specificity as attachment and infection. Moreover, several rhizobial lectin receptors
show biological activity in legume roots (e.g., 5). A possible role for lectin receptors as growth signals will be
discussed, together with the perspectives for the use of the Rhizobium-legame association as a model system
for the study of plant growth at the molecular level,
m DAZZO, F. B. & TRUCHET, G. L. (1983). /. Membrane Biol. 73, 1-16.
(2) QUISPEL, A. et al. (1984) in: Advances in Nitrogen Fixation Research (eds Veeger & Newton) Pudoc,
381-388.
(3) BHUVANESWARI, T. V. see ref. 2, 389-396.
(4) SOLHEIM, B., see ref. 2, 429.
(5) VAN DER HAVE, J. C , see ref 2, 431.
Morphological and biochemical studies on a revertable albino mutant line
olNicotiana tabacum L.cv. Xanthi
A. Kumar, P.K. Evans, P.M.Jordan, Departments of Biochemistry and Biology, University of
Southampton, Southampton S09 3TU
Albinism due to the presence of defective plastids in leaves lacking chlorophyll and other related pigments
is probably the most common and distinct deviation from the wild type amongst the visible mutants in green
plants. This type of mutant has been very useful for studying plastid development, the structural
requirements for photosynthesis and for establishing de novo origin of shoots. We have carried out similar
types of studies using an albino line of Nicotiana tabacum L.cv. Xanthi which initially arose as a spontaneous
mutation that resulted in a variegated leaf and from this albino, callus and shoots have subsequently been
maintained in vitro. The albino line has a tendency to revert spontaneously and randomly to the wild type at
high frequency resulting in a mosaic pattern of variegation in the leaf. This has allowed us to make a
comparative study of the wild type albino and the revertant leaf tissues at structural and biochemical levels.
Histological analysis of revertant leaf materials indicated that the reversion of the albino plastid to the wild
type could occur in all the three layers (LI, LII, LIII) of the apical meristem either separately or together.
Ultrastructure studies of albino plastids show an absence of thylakoid formation and the presence of large
vacuoles. Plastids of revertant leaf material showed an almost similar ultrastructure to the wild type. The gel
profile of the total leaf protein of the albino showed the absence of several bands present in the wild type,
including Fraction 1 protein. The gel profile for revertant total leaf protein was similar to the wild type
although certain bands were missing and these are currently being characterised. The early enzymes of
tetrapyrrole biosynthesis have been measured in the albino and although somewhat lower in activity
compared with the wild type grown under similar conditions, are much higher than that required for haem
synthesis alone. Full results and discussions will be provided.
Plant development
Competence and pattern formation in plant development
19
H. Mohr*, Biological Institute II University of Freiburg, D-78 Freiburg, West Germany
In higher plants light affects ontogeny specifically insofar as the genes which control development can
only express themselves fully in the presence of light (photomorphogenesis). The light effective in
photomorphogenesis is absorbed by sensor pigment phytochrome (P). A physiologically inactive form of P,
Pr, is transformed by light into Pfr, the effector molecule. P is already detectable in the seed, and it is
probable that P is available in every cell of a higher plant during the whole of development. - In
developmental biology 'competence' means that a cell or a tissue is able to respond to a specific (inductive)
stimulus with a specific response. In photomorphogenesis, it is well established that (1) different cells and
tissues can respond differently to Pfr (multiple responses to Pfr) indicating a polytropy of Pfr action (or,
expressed differently, a specifity of 'competence'), and (2) that appearance and disappearance of
competence can best be described in terms of spatial and temporal patterns of competence. Moreover, in
photomorphogenesis pattern specification (appearance of specific competence to Pfr) and pattern
realization (polytropic action of Pfr) can be kept apart. The specification of the temporal and spatial pattern
of competence is independent of light (Pfr) even though the realization of the developmental pattern can
only occur in the presence of Pfr. An example is phy Hot axis, the specific arrangement of leaf primordia on
the apex of growing shoots. There is a strong effect of light, exerted through Pfr, on the rate of formation of
primordia without any interference with the specification of the phyllotactic pattern. Thus, light accelerates
the development of an apex without affecting the temporal and spatial coordination of the events. In some
cases, such as light-mediated flavonoid synthesis or plastidogenesis in angiosperms the molecular mechanism of pattern realization has been elucidated on the level of transcription and enzyme synthesis.
Evidence was obtained that Pfr can modulate as well as induce gene expression once the particular
competence point has been reached in course of development. Present research aims at the molecular
mechanism of pattern specification and concomitant appearance of competence towards Pfr.
Induction versus modulation in phytochrome-regulated gene expression
R. Oelmuller* andH. Mohr, Biological Institute II University of Freiburg, D-78 Freiburg
In higher plants light is required as a signal for normal development (photomorphogenesis). Phytochrome
(P) is the photoreceptor (sensor pigment) involved in photomorphogenesis. The primary action of light is to
transform inactive P (Pr) into physiologically active P (Pfr). Regarding the action of Pfr in photomorphogenesis, evidence has been obtained that Pfr causes stimulation or suppression in the expression of
genes. The lasting question has been whether Pfr causes a modulation of gene expression only or switches
genes on and off. Owing to the difficulties in obtaining precise data with gene-specific hybridisation probes
the question remains undecided as yet. Under these circumstances, we have decided to investigate the
question of whether phytochrome brings about photomorphogenesis through modulation or through
induction of processes at higher levels of gene expression. Products were chosen which can be assayed
precisely and unambiguously. The time course of appearance of competence towards phytochrome (Pfr)
was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light-mediated formation of
anthocyanin (aglycone cyanidin) and NADP-dependent plastidal glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13). The experiments were performed to answer the following question: Does
phytochrome act to turn these responses on (induction), or, as an alternative, does phytochrome cause an
amplification of processes already occurring in absolute darkness albeit at low rates once competence is
reached (modulation)? The data show that in the case of GPD, phytochrome causes an amplification of the
rate of synthesis once the competence point is reached at approximately 36 h after sowing at 25 °C. In the
case of 'early anthocyanin' (competence point at 26 h), synthesis does not occur in darkness without Pfr,
while in the case of 'late anthocyanin' (competence point at 39 h), phytochrome causes an amplification of a
process occurring in complete darkness albeit at a very low rate. It is concluded that Pfr mediates
photomorphogenesis by modulation as well as by induction of gene expression.
20
Plant development
Cell lineage patterns in maize development
R. S. Poethig*, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
The cellular parameters of shoot development in maize were characterized by using x-ray-induced somatic
sectors to mark cell lineages (clonal analysis). Sectors generated at various stages in embryogeny
demonstrate that there is a progressive increase in the number of cells that ultimately form the shoot apex.
Just prior to its differentiation, the shoot apical meristem encompasses about 100 cells in the paradermal
plane of the embryo, and extends inward at least 2 cell layers. The developmental fate of shoot initial cells
depends on their position in the meristem. Peripheral cells form the basal nodes of the shoot, whereas more
centrally located cells contribute solely to upper nodes. Although nodes become clonally isolated from one
another during the development of the shoot, there is no evidence that cell lineages obey strict compartment
boundaries. In fact, the orientation and extent of cell division is quite variable at any stage in development,
and in any given part of the shoot.
After germination the shoot meristem lays down a series of more-or-less identical vegetative segments
before it differentiates into a tassel. Clonal analysis demonstrates that the leaf is clonally related to the
intemode and the axillary bud below it, rather than to the internode and bud that it subtends (Johri and
Coe; Dev. Biol. 97: 154-172, 1983). Further observations on the cell lineage of the leaf and internode and
on the pattern of cell division during leaf development will be presented.
Perithecial neck development in Sordaria humana
N. D. Read, Department of Botany, University of Bristol, Bristol BS81UG
Perithecial neck development has been studied in the ascomycete fungus Sordaria humana using a variety
of light and electron microscope techniques. The developing neck is composed of two basic cellular types
(Read, 1983): (i) discrete hyphal-like periphyses which line the central neck canal, and which are
surrounded by (ii) coherent peridial cells which comprise the neck peridium. The periphyses are relatively
undifferentiated and exhibit a pronounced longitudinal growth pattern. The peridial cells show varying
degrees of differentiation and can only grow by expansion and secondary cell wall thickening. Neck
development is indeterminate. Extension occurs primarily at the neck apex and involves the differentiation
of periphyses into the neck peridium. This process involves the differentiation of periphyses by a 'cohesive
cement'; secondary wall thickening; melanization; and finally cell autolysis. The periphyses are bathed in
mucilage. Components of the mucilage may be involved in the formation of the cohesive cement. The
modes of septal pore occlusion differ between the periphyses and neck peridium. The neck is positively
phototropic and its curvature results from the differential growth of periphyses. Some of the basic structural
features of fungal multicellular development exhibited during perithecial neck development have been
compared with the modes of development found in other organisms.
READ, N. D. (1983). A scanning electron microscopic study of the external features of perithecial
development in Sordaria humana. Can. J. Bot. 61: 3217-3229.
Plant development
Controls of leaf shape
21
Tsvi Sachs*, Department of Botany, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
The varied shapes of leaves result from differential oriented growth of similar primordia. These primordia
resemble regulative embryos in their capacity to regenerate following severe damage. Their development
requires no external cues but does depend on species, point mutations, environmental conditions and state
of the plant. The general questions to be considered are those of pattern determination: how are the
different fates of the parts of primordium specified and how is shape, as distinct from size, modified by
factors that act on the entire developing leaf.
Leaf development starts by determinate primordial growth along one major axis. Primordia formation
may be repeated from the flanks of the original primordium, building the framework of the leaf and the
pattern of the major veins. The extent of the original primordial growth can be specifically influenced by the
state of the plant and by growth factors known to originate in the roots, and this is expressed by the number
of lateral centers of growth which develop as leaflets or lobes. One control of shape is thus the specific
sensitivity of an early, defined developmental process.
The expansion of leaf tissues is either in one dimension, forming axial structures, or in two dimensions,
forming the flat surface of the lamina. The relative role, timing and location of these two modes of
expansion are major determinants of leaf shape. Auxin, known to be formed in developing leaves, induces
unusual axial growth when applied repeatedly, suggesting a mechanism by which axes are induced by the
lamina. Spatial correlations may thus determine differences between parts of the leaf. Temporary auxin
application, on the other hand, causes lamina growth that connects leaflets and even individual leaves.
These major shape modifications could be due to changes of the timing of expansion which results in the
growth of cells that would otherwise lose the competence to expand before the right conditions occur.
These and additional results suggest how quantitative differences of basic developmental processes could
account for leaf shapes in mutant peas and in species of Lathyrus. They also suggest how special
developmental programs can be controlled in parts of leaves, leading, for example, to the formation of
tendrils, and how these programs can be switched by 'homeotic' mutations.
Sexual differentiation in Chlamydomonas eugametos
H. van den Ende*, A. Musgrave, A. Tomson andR. Demets, Department of Plant Physiology, University of
Amsterdam, Kruislaan318,1098 SM Amsterdam, The Netherlands
Chlamydomonas is an unicellular green alga with twoflagella.Sexual reproduction between isogametes of
opposite mating type (mt+ and mt~) is initiated by mutual adhesion of the flagella. This adhesion displays
extreme mating-type specificity. It triggers the activation of a special zone of the plasma membrane where
subsequently fusion between the mates occurs. We have isolated a high-molecular weight hydroxyprolinerich arabinogalactan glycoprotein from the flagellar surface of C. eugametos, called the mt~-agglutination
factor, which mediates the flagellar adhesion. It is also present on the plasma membrane of gametic cell
bodies, and we don't understand why. We present evidence that there is no continuity between the plasma
membrane and theflagellarmembrane with respect to the mt~-agglutination factor. During gametogenesis,
the mt~-agglutination factor first appears in a cellular compartment, probably the plasma membrane, and
subsequently at theflagellarsurface. The activity and/or presence of the agglutination factor at the flagellar
surface, but not in the cellular compartment, is subject to the light/dark regime. This results in a fluctuating
mating competence of the gametes.
22
Plant development
Positional information and pattern formation in plants: abscission zones
P. M. Warren Wilson and J. Warren Wilson, Departments of Developmental Biology and Botany, Australian
National University, Canberra, ACT26O1, Australia
Abscission of leaves, fruits and other plant parts occurs by the differentiation of transverse 'separation
layers' across leaf stalks, fruit stalks and other organs. This study explores the mechanisms defining the
position of such abscission zones, by exploiting certain abscission phenomena in Impatiens sultani.
When short (10-30 mm) segments cut from Impatiens stems are cultured aseptically, most of these
explants - though initially uniform longitudinally - develop a transverse separation layer after 1-2 weeks,
and the top then abscises from the bottom. The distance of the separation layer from the base of the explant
does not vary with explant length, but increases with the concentration of auxin (indole acetic acid) applied
basally.
Results from these internodal stem explants, from surgical operations on shoots, and from experimental
applications of auxin, suggest that abscission zone positioning is controlled primarily by auxin acting as a
morphogen; abscission sites occur at Y-junctions just above the base of the arm with lower activity and
auxin status, or in single axes above a region of higher auxin status. Both sites are distinguished by the auxin
concentration decreasing in the apical direction (Warren Wilson, 1984).
This hypothesis is supported by a mathematical model of the interaction of diffusive transport and active
basipetal polar transport of auxin in controlling the concentration gradient along internodes with specified
auxin concentrations maintained basally. The model allows predictions concerning the site and timing of
abscission which accord with observations on internodal explants, and these relations yield acceptable
estimates of the two transport coefficients.
WARREN WILSON, J. & P. M. (1984). Control of tissue patterns in normal development and in regeneration.
In Positional Controls in Plant Development (ed. P. W. Barlow & D. J. Carr), pp. 225-80. Cambridge
University Press.
Positional information and pattern formation in plants: vascular tissues
J. Warren Wilson and P. M. Warren Wilson, Departments of Botany and Developmental Biology, Australian
National University, Canberra, ACT2601, Australia
The vascular tissue in a dicotyledon stem first develops as a hollow cylinder of procambium, which forms
phloem on the outside and xylem on the inside, with the dividing cambial layer between them. If the stem is
wounded so as to interrupt this vascular cylinder, cell division yields a wound callus within which a new
cambium regenerates, uniting the cut ends of the original cambium and forming phloem outwards and xylem
inwards; this restores the vascular cylinder.
The gradient induction hypothesis (Warren Wilson, 1984) states that cambial positioning is controlled
both by a surface effect, such that a cambium arises a certain distance beneath any exposed surface, and also
by a homeogenetic inductive effect of differentiated vascular tissues. These effects are mediated through the
concentrations of two morphogens, auxin and sucrose: the surface effect arises through auxin destruction at
exposed surfaces, and the inductive effect through the diffusion of auxin and sucrose from xylem and
phloem respectively. The concentrations of auxin and sucrose are maintained in these two sources, and
opposed gradients of these two morphogens occur across the cambium. A cambium can form in
undifferentiated tissue only where the auxin/sucrose concentration ratio equals that at the original cambium;
it forms phloem and xylem towards lower and higher values of the ratio respectively.
These interpretations are supported by evidence on auxin and sucrose concentration gradients across the
cambium, and on their sources, movements, and known effects on vascular differentiation. Simulations of
the hypothetical positional control system predict vascular patterns corresponding to those observed in four
types of wound and graft, and after experimental auxin application.
WARREN WILSON, J. & P. M. (1984). Control of tissue patterns in normal development and in regeneration.
In Positional Controls in Plant Development (ed. P. W. Barlow & D. J. Carr), pp. 225-80. Cambridge
University Press.