Table Of ContentPLANT ROOTS - FROM CELLS TO SYSTEMS
Developments in Plant and Soil Sciences
VOLUME 73
The titles published in this series are listed at the end of this volume.
Plant Roots - From Cells to Systems
Proceedings of the 14th Long Ashton International Symposium Plant Roots -
From Cells to Systems, held in Bristol, U.K., 13-15 September 1995
Edited by
H. M. ANDERSON, P. W. BARLOW, D. T. CLARKSON, M. B. JACKSON
AND P. R. SHEWRY
Partly reprinted from Plant and Soi/, Volume 187, No. 1 (1996)
SPRINGER SCIENCE+BUSINESS MEDIA, B.V.
Library ofCongress Cataloging-in-Publication Data
ISBN 978-94-010-6402-6 ISBN 978-94-011-5696-7 (eBook)
DOI 10.1007/978-94-011-5696-7
Printed on acid-free pap er
Ali rights reserved
© 1997 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 1997
Softcover reprint of the hardcover 1s t edition 2004
No part of the material protected by this copyright notice may be reproduced
or utilized in any form or by any means, electronic or mechanical,
including photocopying, recording or by any information storage and
retrieval system, without written permission from the copyright owner.
Contents
*
1. Mutational analysis of root initiation in the Arabidopsis embryo
T. Berleth, C.S. Hardtke, G.K.H. Przemeck, J. Muller
*2. Turgor-regulation during extension growth and osmotic stress of maize roots. An example of
single-cell mapping
J. Pritchard, W. Fricke, D. Tomos 11
*3. Microtubular cytoskeleton and root morphogenesis
P.w. Barlow, J.S. Parker 23
*4. Roots of willow (Salix viminalis L.) show marked tolerance to oxygen shortage in flooded soils
and in solution culture
M.B. Jackson, P.A. Attwood 37
*5. Rol genes and root initiation and development
D. Cbriqui, A. Guivarc 'h, W. Dewitte, E. Prinsen, H. van Onkelen 47
*6. Variation, co-ordination and compensation in root systems in relation to soil variability
D. Robinson 57
*7. Water transport in plants: Role of the apoplast
E. Steudle, J. Frensch 67
*8. The molecular basis of potassium nutrition in plants
c.J. Smart, D.E Garvin, J.P. Prince, W.J. Lucas, L.V. Kochian 81
*9. Root apical organization in Arabidopsis thaliana ecotype 'ws' and a comment on root cap
structure
T.L. Rost, S.E Baum, S. Nichol 91
*
10. Experimental and genetic analysis of root development in Arabidopsis thaliana
B. Scheres, H. McKhann, C. van den Berg, V. Willemsen, H. Wolkenfelt, G. de Vrieze, P.
Weisbeek 97
11. A biophysical analysis of root growth under mechanical stress
A.G. Bengough, C. Croser, J. Pritchard 107
12. Metal-gene-interactions in roots: metallothionein-like genes and iron reductases
N.T. Robinson, J.R. Wilson, J.S. Turner, A.P. Fordham-Skelton, Q.J. Groom 117
13. Molecular responses of roots related to fungal colonisation in arbuscular mycorrhiza
V. Gianinazzi-Pearson 131
• Chapters indicated with an asterisk are reprinted from Plant and Soil Volume 187, No.1 (1996)
14. Legume and actinorhizal root nodule formation
K. Pawlowski, T. Bisseling 137
15. Responses of roots to low temperatures and nitrogen forms
P. Stamp, B. Feil, M. Schortemeyer, W. Richner 143
P!([1lI ([lid SlIi! 187: 1-9, 1996.
© 1996 Kiliwer Academic Publishers.
Mutational analysis of root initiation in the Arabidopsis embryo
Thomas Berleth, Christian S. Hardtke, Gerhard K.H. Przemeck and Judith Muller
Genetics Institute, Univ. Munich (LMU), Maria-Ward-Str. la, D80638 Munich, Germany*
Received 16 January 1996. Accepted in revised form 26 April 1996
Key words: Arabidopsis , cell axialization, embryo, root, vascular tissues
Abstract
In dicoty ledonous plants, primordia of most seedling organs are laid down between fertilization and the formation
of the heart-stage embryo. In Arabidopsis embryogenesis, highly regular cell divisions and cell expansions facilitate
the characterization of mutant development.
We have taken a genetic approach to identify genes involved in organising the hypocotyl/root axis. The initiation
of this axis is marked by oriented expansions and longitudinal divisions of cells in the lower tier of the early
globular embryo. These cells go on to form a defined number of parallel cell files that constitute the hypocotyl and
most of the radicle. Mutants impaired in the initiation or elaboration of the hypocotyl/root axis were selected by
their seedling phenotype and subsequently analysed at embryonic stages.
Several conclusions are suggested by the phenotypes of these mutants. First, hypocotyl/root axis formation can
be genetically separated from other aspects of embryonic pattern formation. Second, initiation of the hypocotyl/root
axis appears to be genetically distinct from post-embryonic root initiation. Third, four loci were identified that
appear to contribute to the elaboration of the axial pattern. Finally, an anatomical inspection of one of the mutants,
amenable to an analysis at post-embryonic stages, suggests a genetic link between basal pattern formation in the
embryo and aspects of vascular differentiation in the adult plant.
Introduction distortions, while on the other hand a high degree of
reproducibility facilitates the identification of mutants.
In most organisms the molecular mechanisms directing Furthermore, structural simplicity should be accom
cells towards specific fates are still elusive. Although it panied by a multitude of recognizable markers and
is generally accepted that this specification results from finally, mutant screens can only be performed in few,
hierarchically organized gene activities, the respective genetically accessible species.
genes and the mechanisms of control have been identi By these criteria, the Arabidopsis root (as well as
fied only in few exceptional cases. In suitable systems, the hypocotyl) seems particularly suited for mutant
large-scale screens for embryonic mutants (followed analysis. The structure of the primary root is highly
by mutant characterization and the isolation of the reproducible in the wild type, but even gross distor
identified genes) have been performed and shown to tions are compatible with some extent of root growth
provide access to a molecular analysis of developmen ( Aeschbacher et aI., 1995; Baskin et aI., 1992; Ben
tal processes (reviewed by Ingham, 1988; Schnabel, fey et a!., 1993; Scheres et a!., 1995). The anatom
1991). Successful mutant analysis appears to be the ical structure is simple and easy to analyse, but still
crucial step in this strategy, and only few developmen bears numerous recognizable landmarks (Ben fey and
tal processes seem to be amenable to genetic analysis. Schiefelbein, 1991, 1994). The anatomical organisa
This might be, because the developmental process has tion of the embryo- derived structures, the hypocotyl
to meet conflicting requirements. It should be flexi and the primary root, has been described in detail
ble in order not to be arrested by mutation-induced (Dolan et a!., 1993), and the developmental origin of
the hypocotyl/root axis has been investigated usmg
* FAX No: +498917919820
2
genetic cell lineage markers (Scheres et aI., 1994). allel elongations and oriented divisions of descendants
Finally, Arabidopsis is considered to be a favorable of the !.t. cells endow the embryo with an anatomical
organism for genetic analyses of developmental pro ly visible body axis (Figures lC,D). The cell files of
cesses in higher plants (Meyerowitz, 1989). this axis are attached to the uppermost suspensor cell
Root meristems are established throughout plant ("hypophysis cell"), which becomes distinct from the
development. In the embryo of dicotyledonous plants, remaining suspensor cells upon undergoing a highly
oriented divisions of basally located cells give rise to an stereotyped division pattern. In these divisions a cell of
integrated axial structure encompassing the hypocotyl unique shape ("lens-shaped" cell, Figures ID,E) is gen
and the radicle. Due to this developmental origin, erated and prominent cell walls separate the hypoph
hypocotyl and radicle are often difficult to delimit, ysis descendants from the /.t. descendants throughout
although they differ in ontogeny, anatomy and some embryo development (Figures IE-H). Descendants of
physiological responses. For clarity in mutant descrip the "lower tier" cells undergo highly uniform division
tions, we will use the term hypocotyllroot axis when patterns to produce a nearly constant number of cell
referring to the entire axial structure in the embryo, files, including centrally located narrow procambial
and the term hypocotyl to refer to the region defined cells (Figures IH). An additional periclinal division
by two layers of cortex cells, thus also covering an splits the cortex layer at an early stage (Figures IE-H),
"intermediate zone" giving rise to some root features resulting in two layers of cortex cells along most of the
(Scheres et aI., 1994). hypocotyl/root axis.
In the following we will give a short description Cell division patterns may differ considerably
of Arabidopsis embryogenesis with particular empha between embryos of different dicot species or may not
sis on the formation of the hypocotyl/root axis and be regular at all (Johri, 1984). Irregular cell divisions
describe developmental defects in four mutants affect have also been observed in Arabidopsis mutants that
ed at successive steps in embryonic hypocotyllroot axis still form normal seedling patterns (Torres and Jiirgens,
formation. 1994; Traas et aI., 1995; Przemeck, unpubl.). Thus
underlying molecular mechanisms of embryo pattern
formation are not likely to act through ordered cell divi
The formation of the hypocotyVroot axis in the sions, yet a stereotyped development may allow for the
Arabidopsis embryo recognition and classification of deviations from the
normal course of embryogenesis.
As a Crucifer, Arabidopsis follows the onagrad type of The scheme in Figure 2 illustrates an interpretation
embryogenesis which is characterized by a high degree of stages shown in Figure 1. Groups of cells that appear
of reproducibility (for detailed descriptions and ter to embark on specific developmental pathways, recog
minology of onagrad embryogenesis see Johri, 1984; nized by defining characteristics of cell shape and/or
Mansfield and Briarty, 1991; Tykarska, 1976, 1979). division pattern, are marked by different shadings. In
Figure I shows a series of eight characteristic stages, this schematic view, the initiation of the hypocotyl/root
each characterized by specific behaviour of cells at axis can be considered as the third of four successive
particular sites. Early divisions (not shown) generate pattern forming processes in the early embryo (outlined
two cell populations, one derived from the basal and below); it remains to be seen whether these processes
the other from the apical daughter cell of the zygote can be separated genetically.
(descendants referred to as "a" and "b" cells in the First, the descendants of the apical and basal prod
following). These form the filamentous suspensor and ucts of the first zygotic division can be distinguished by
the "embryo proper", respectively (Figure lA). While differences in the orientations of subsequent cell divi
the number of suspensor cells is somewhat variable, sions: The plane of successive divisions is shifted at a
the cell divisions in the "embryo proper" are highly maximum angle in the former while remaining fixed in
reproducible, generating a symmetrical arrangement of the latter (Figure 2A). Second, tangential divisions at
eight cells ("octant" stage), arranged in an "upper" and the octant stage give off a layer of protoderm cells that
a "lower tier" of four cells each (u.t .• !.t., Berleth and are characterized by anticlinal divisions (Figure 2B).
JUrgens, 1993). Following the" "octant" stage, tangen In a third major pattern forming step the inner cells of
tial divisions give rise to a protoderm layer ("dermato the dermatogen-stage embryo become subdivided into
gen" stage embryo, Figure lB). The embryo remains isodiametric u.t. and elongating l.t. cells (Figure 2C)
globular during the subsequent cell divisions, but par- and shortly afterwards, the hypophysis cell becomes
3
,
c
( F I
Figure 1. Selected stages of Arabidopsis embryogenesis (structures are marked in Figure 2). (A) Octant stage: the cells of the embryo proper
stain darkly. (B) Dermatogen stage: a tangential division of each of the eight cells of the "octant" produces inner-and epidermis cells. (C. D)
Globular stages: cells of the upper and lower tier of the inner cells elongate differentially (C) and cell shape differences between cortex and
procambial cells become apparent (D). (E) Early heart stage: embryo shows primordia of all major pattern elements (compare Fig. 2). Central
vascular primordium in the hypocotyllroot axis and single and double cell-layers in the cortex are visible. The hypophysis cell (uppermost
suspensor cell) has undergone one horizontal division. (F) Late-heart stage: hypophysis derivatives have formed a characteristic assemblage
of twelve cells arranged in three tiers (six visible in the section). Hypocotyl and cotyledons are enlarged. (G) Mid-torpedo stage: Further
enlargement of hypocotyl and cotyledons. root cap is clearly established. Vascular differentiation in the hypocotyl extending into the cotyledons.
(H) Mature embryo: clear demarcation of the vascular cylinder. Note that hypophysis descendants are still clearly recognizable. The double-cell
layered cortex region is approximately 25 cortex cells in length. (A. E. G reproduced from Berleth and Jurgens. 1993).
distinct from the remaining suspensor cells by under hypocotyl and root are not essential for embryonic via
going a series of precisely defined divisions (not visible bility or germination (Jurgens et aI., 1991; Mayer et
in Figure 2). l.t.- and hypophysis-cell descendants are aI., 1991). Thus, mutants affected in these structures
both distinct from their apical and basal neighbours should be recognizable at the seedling stage. While
and contribute to an integrated seedling structure, the many root mutants can only be identified after growth
hypocotyl/root axis. In a fourth step, cotyledon pri on sugar containing media, root initiation mutants can
mordia emerge due to increased mitotic activity of be recognized immediately after germination on water
U.t. descendants at two opposite positions (Figure 2D). agar. As this obviates the need for seed sterilization,
For clarity, mutant phenotypes described in subsequent screening for potentially lethal phenotypes in thou
sections will be related to this scheme. sands of individual lines becomes feasible. Figures 3
and 4 show examples of mutant phenotypes affected at
three stages of hypocotyllroot axis formation. Mutants
Mutations affecting primary root formation will be interpreted by describing their phenotypes in
the terms of the scheme of Figure 2.
A number of seedling phenotypes obtained in large Seedlings mutant for the gene STUMP (STP)
scale genetic screens demonstrate that formation of resemble wild-type Arabidopsis seedlings, but fail to
4
c
A B D
Figure 2. Scheme of Arabidopsis embryogenesis. (A) Octant-, (B) dermatogen, (C) early globular, (D) early heart stage. Schemes are drawn
according to the corresponding stages in Figure I. Cell populations distinguishable by cell shape and division pattern are marked by differential
shading. Marked pattern elements: (sus) supensor, (ep) epidermis, (hy) hypocotyl, (crm) centre of root meristem, (ra) radicle, (vp) vascular
primordium, (co) cotyledon. Details are explained in the text.
Figure 3. Selected stages of wild-type and mutant development. (A, I) Arabidopsis wild-type at the seedling stage and at early heart stage
of embryogenesis (arrowhead in I marks hypophysis derivatives). (B, C) Seedling phenotype of the sip mutant: in contrast to mw and bad
mutants, hypocotyls are of normal length (B) and end in morphologically normal root meristems (C). (D, E) Sections of mature embryos from
the progeny of SIp heterozygous plants do not reveal anatomical abnormalities in longitudinal sections of the radicle (E) or cross sections
through the hypocotyl (D). (F, G) Bad mutant seedlings. Hypocotyl is reduced in length and ends basally in apparently not differentiated cells.
Vascular system in the cotyledons appears only slightly affected (G). (H) mw mutant embryo at early heart stage displaying enlarged hypophysis
derivatives and abnormal planes of cell division (arrowhead), while more apical regions in the pro-embryo appear not severely distorted.
