New Phytol.

(1997), 137, 1-8

Genetic approaches in plant physiology
BY M. KOORNNEEF*, C. ALONSO- BLANCO AND A. J. M. PEETERS
Department of Genetics, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA,
Wageningen, The Netherlands
{Received 3 April 1997; accepted 24 June 1997)
SUMMARY
The use of genetics in plant biology aims at the physiological and molecular genetical characterization of the
phenotypic variation for the trait under study. Efficient mutant and gene isolation procedures have been developed
for a number of plant models such as Arabidopsis thaliana. For this, the map position of the genes and insertion
mutagenesis are used. The latter also allows the characterization of genes that are not easily recognized in mutant
approaches, by using enhancer or gene-trapping procedures and reverse genetics. In addition to mutants, natural
variation present among wild and cultivated varieties of a species provides an important source of genetic variation.
The use of molecular markers, advanced mapping populations and specific cytogenetic stocks in case of polyploids,
enables a detailed characterization of such natural variation even when it is of a quantitative and polygenic nature.
Examples of the various genetic approaches are given.
Key words: Arabidopsis thaliana, abscisic acid (ABA), gene isolation and mutagenesis, photomorphogenesis,
quantitative trait locus (QTL).
INTRODUCTION
The use of genetics as a tool to dissect complex
biological processes in plants has a long history. For
instance, a tobacco mutant called Maryland Mam-
moth and different soybean varieties led to the
discover}' of photoperiodism by Garner & Allard
(1920). Later, during the fifties, genetic dwarfs in
pea and maize convinced many of the importance of
gibberellins as plant growth hormones. Why there-
after genetics has not been used very much in plant
physiology is less clear, despite that mutants were
shown to be crucial tools for the understanding of
biosynthetic pathways in micro-organisms and de-
velopmental processes, e.g. in Drosophila. Towards
the end of the seventies, students of E. coli and
Drosophila genetics such as Somerville and Meyer-
owitz became convinced that genetics was the way to
go ahead in plant science. These authors, as well as
Laibach, Redei and Feenstra before them, had
realized that Arabidopsis thaliana (L.) Heynh. was a
model species for plant genetics, because this small
self-fertilizing crucifer has a very short generation
time. Genetics becomes even more powerful when it
can be combined with molecular genetics, which
links DNA with the phenotype. Its small genome
and ease of transformation were additional factors.
* To whom correspondence should be addressed.
E-mail: maarten. [email protected] .wau .nl
which established Arabidopsis as the general model
organism in higher plant molecular genetics. Cur-
rently, the whole genome of this plant is being
sequenced (Bevan et al.., 1997), and the impact of this
information is rapidly increasing as can be seen, for
example, from the frequent use that is already made
of the partly sequenced cDNAs called expressed
sequence tags (ESTs).
Besides Arabidopsis, other plant species such as
petunia. Antirrhinum, maize, pea, tomato, barley and
rice have been studied for a long time, resulting in
large collections of genetic stocks and genetic maps.
They became very interesting genetic models for
specific developmental processes because of the
particular characteristics of these species, which led
to the characterization of some of their mutants. For
instance, for Petunia and Antirrhinum, mutations
affecting flower colour and morphology were
analysed in detail. With the advent of molecular
biology. Antirrhinum, maize and, somewhat later.
Petunia could be used for gene cloning, since well
characterized transposable elements were available.
Unfortunately, these species, except Petunia, are
difficult or impossible to transform, which limits the
complementation proof of having cloned the gene
and the further characterization of those genes.
Tomato and, even more so, rice, have relatively
small genomes, allowing the application of map-
based cloning. Furthermore, maize transposable
elements have been introduced into these species.
M. Koornneef, C. Alonso-Blanco and A. J. M. Peeters
resulting in the isolation of several disease-resistance
genes (Jones & Jones, 1997). The larger genomes of
barley and pea make these classic model species less
amenable to some molecular approaches. However,
the value of these species should not be under-
estimated, given their suitability not only for genetics
but also for plant physiology and plant biochemistry.
In pea, the system of nitrogen fixation could be
studied genetically, and well characterized mutants,
e.g. in the phytochrome, flowering and gibberellin
pathways, resulted in excellent research, comple-
mentary to that of the ' general' models. In barley,
similar approaches provided important molecular
data on seed germination, and the recent map-based
cloning of the mildew resistance gene Mlo (Blischges
et al, 1997) indicates that these limitations, because
of the large genome, can be overcome. Thus, it is
clear that genetics nowadays is a basic tool in plant
science. In order to apply this genetic approach,
genetic variation, which takes a range of forms, is
essential. The generation of mutant variation is
generally not a problem because efficient mutagens
and mutagenesis procedures are available. More
difficult is the identification of mutant phenotypes
that are relevant to the research topic. For mor-
phological processes the phenot>^pe itself allows the
detection of mutants; however, for some biochemical
and physiological traits, either the phenotype is
relatively subtle, or the phenotype is too general, e.g.
general reduction of plant size or vigour. An
important limitation in finding mutants is that for
many genes redundancy is present, which means that
mutations in such genes do not result in an obvious
visible phenotype since the redundant counterpart
produces enough product to (at least partly) sub-
stitute for the function of the mutated gene. In
addition to induced mutants, another source of
genetic variation is provided by the natural variants
present within a species and by cytogenetic stocks
such as chromosomal substitutions. Once genetic
variation has been identified and characterized, the
application of molecular genetic techniques to amen-
able species allows the cloning of the mutated genes.
Cloning and characterization of genes have some-
times given clues about the nature of the observed
defects in the mutants. An important recent example
is the identification of Arabidopsis and tomato genes
producing dwarfism when mutated, in which en-
coded proteins are related to steroid biosynthesis
proteins in mammals (Li et al, 1996). This prompted
several authors to test the effect of brassinosteroids
on these dwarf mutants, of which many could be
reverted to the phenotype of the wild type, whereas
others were classified as insensitive to this group of
compounds (Kauschmann et al,\996). Brassinos-
teroid mutants have common characteristics, such as
being dwarf with small round leaves and having a de-
etiolated phenotype when grown in darkness. These
findings convincingly showed the importance of
brassinosteroids as natural plant hormones. On the
other hand, it is very important to find an effect on
the phenotype by genes that have been cloned and
sequenced but for which no mutant phenotype is
known, since such a phenotype provides key in-
formation about the function of those genes. New
approaches such as reverse genetics are being
developed to generate mutants, starting with a DNA
sequence of unknown function.
THE USE OF MUTANTS TO CLONE GENES AND
THE USE OF GENES TO ISOLATE MUTANTS
Anumber of procedures are now available to isolate
the corresponding DNA of genes only known by
their mutant phenotype. The most important of
these methods are gene tagging and map-based
cloning.
1. Gene tagging
Insertion of DNA in a coding sequence in many
cases disrupts the gene, resulting in a mutant
phenotype. Since the sequence of the inserted DNA
is known, it is possible to clone the genomic plant
DNAflanking the insertion and thereby part of the
disrupted gene. In plants, T- DNA and transposable
elements are most frequently used for tagging. In
Arabidopsis, several large collections of T- DNA
insertions have been generated (Feldmann, 1991)
and, more recently, the introduction of maize
transposable elements such as the Ac/ Ds (Bancroft
et al.,1992) and the En/ I (Aarts et al, 1995) two-
element systems have been used successfully.
A specific application of tagging is the use of
enhancer or promotor traps (Sundaresan, 1996). In
this system the T- DNA or transposable element
carries a reporter gene without a promotor or with a
minimal promotor. When such a construct is inserted
near to a gene the expression pattern of the reporter
gene might reflect the expression pattern of the
endogenous gene, whose promotor or enhancer was
used to drive the expression of the reporter gene.
Because of redundancy, and also because certain
functions might not be essential, such insertions do
not always result in physiological and morphological
mutant phenotypes. Very sophisticated systems of
this type, which include gene traps, have been
developed recently (Sundaresan et al, 1995). Such
trap lines have been shown to be very useful for
marking specific tissues (Scheres et al, 1994) or
processes. An important novel application of tagging
is in reverse genetics (Koes et al, 1995). Plants with
insertions in cloned genes, for which some sequence
information is available, can be identified by the
ability to amplify DNA fragments m polymerase
chain reaction (PCR). One PCR primer is based on
the tagging-DNA, whereas the second is based on
the sequence for which the insertion mutant is
Genetics in plant physiology
sought. Again a mutant phenotype will not always be
observed when the respective gene is disrupted,
because of redundancy.
2. Map-based cloning
Map-based cloning requires a detailed genetic map-
position of the locus and the availability of easily
screenable libraries harbouring large plant DNA
fragments, e.g. in yeast and bacterial artificial
chromosomes (YACs and BACs). The availability of
complete physical maps, where such clones have
been grouped and sorted into contigs that are related
to the genetic map are now available for most of the
Arabidopsis genome (Schmidt et al., 1995, Zachgo et
al., 1996). This enormously facilitates map-based
cloning since it restricts chromosome walking to
chromosome landing (Tanksley, Ganal &Martin,
1995). Final proof that the right gene has been
cloned should come from the complementation of
the recessive (mostly mutant) phenotype by DNA
from the genotype with the dominant allele and/or
by sequencing several mutant alleles.
3. Other gene-cloning strategies based on mutants
Additional cloning techniques based on mutants are
genomic subtraction (Sun, Goodman &Ausubel,
1992), which requires deletion mutants, and differ-
ential cDNA display in which PCR or hybridization
screening is performed comparing wild type and
mutant (Sommer et al.,1990).
THE USE OF MUTANTS TODISSECT TRAITS
Mutants have been proved important either for
establishing biochemical pathways or to identify the
physiological role of the mutated genes and pro-
cesses. Cloning of the corresponding genes has often
provided important, and sometimes unique, in-
formation on the mode of action of these genes and,
furthermore, it offers the possibility of modifying
these processes, e.g. by reintroducing the cloned
genes into plants, thereby over-expressing or under-
expressing the genes.
Three different pathways, namely those affecting
abscisic acid (ABA), phytochrome and the transition
to fiowering can serve as illustrations for the genetic
dissection of physiological pathways.
1. Abscisic acid
The plant hormone abscisic acid (ABA) affects many
processes in plants. Its role in controlling seed
dormancy and stomatal closure, together with its
germination and growth-inhibiting effect led to the
isolation of mutants affected in ABA biosynthesis
and action (Table 1). The aba! mutants, which are
defective in epoxy-carotenoid levels and ABA,
provided conclusive proof for the carotenoid path-
way of ABA biosynthesis in higher plants (Duckham,
Linforth &Taylor, 1991; Rock &Zeevaart, 1991).
Marin et al. (1996) cloned this gene using transposon
tagging. The ABA mutants showed the importance
of ABA in stress resistance (reviewed by Giraudat et
al., 1994). Three different genes of ABA-insensitive
{abi) mutants have been cloned. ABIl (Leung et al.,
1994; Meyer, Leube &Grill, 1994) and ABI2
(Leung, Merlot &Giraudat, 1997) were both shown
to encode protein phosphatase 2Cs, which apparently
play a role in ABA signal transduction. The ABI3
gene encodes a seed-specific transcription factor that
transmits the ABA signal, as well as unknown
developmental signals, to a number of seed-develop-
ment-specific genes (Parcy &Giraudat, 1997). The
relevance of ABA in seed germination is shown by
the lack of seed dormancy, characteristic of almost all
ABA related mutants identified so far. Reciprocal
Table 1. Loci involved in the biosynthesis or mode of action of ABA, and phenotype of the mutants at these loci
Gene
Seed*
dormancy
Stomatal
closure*
ABA sensitivity
growth inhibition* Gene function
References
ABAl
ABA2
ABA3
ABIl
ABI2
ABB
ABI4/5
ERAl
ERA2/3
GCAl/8
-t
t
+ t
+ t
Zeaxanthin epoxydase
Conversion of xanthoxin to ABA
aldehyde
Addition of sulphur to Moco of
ABA aldehyde oxidase
Protein phosphatase 2C
Protein phosphatase 2C
Seed-specific transcription factor
/3 subunit of farnesyl transferase
Marin et al. (1996)
Schu^artz et al. (1997)
Schwartz et al. (1997)
Leung et al. (1994);
Meyer et al. (1994)
Leung et al. (1997)
Giraudat et al. (1992)
Finkelstein (1994)
Cutler et al. (1996)
Cutler et al. (1996)
Benning et al. (1996)
* Reduced ( ), similar ( + ) or enhanced (+ +) as compared to wild type,
t At the level of germinating seeds only.
4 M. Koornneef, C. Alonso-Blanco and A.J. M. Peeters
Table 2. Arabidopsis mutants affecting photoreceptive pigments
Sensitivity to*
Gene Bf R FR
Gene function
References
HYl
HY2
HY3 (PHYB)
HY4
HY5
PHYA +
Heme oxygenase J
synthase I

Phytochrome B
Cryptochrome
Transcription factor
Phytochrome A
Parks & Quail (1991); Terry
(1997)
Parks & Quail (1991); Terry
(1997)
Reed et al. (1993)
Ahmad & Cashmore (1993)
Oyama et al. (1996)
Whitelam et al. (1993)
* The sensitivity of mutants at these loci to light of specific wavelengths is less than ( ), slightly less than ( + ) or
the same as ( + ) wild type.
t B, Blue light; R, Red light; FR, Far Red light.
X Heme oxygenase and phytochromobilin synthase control the two last steps of phytochrome chromophore
biosynthesis.
crosses revealed that ABA produced by the embryo
controls germination (Karssen et al., 1983). Without
ABA, seeds do not require gibberellin (GA) for
germination as shown by their resistance to the
gibberellin biosynthesis inhibitors tetcyclacis and
paclobutrazol (Leon-Kloosterziel et al., 1996a, b).
In addition, the insensitivity to such inhibitors of
seed germination led to the isolation of aba2 and
aba3 mutants and to the isolation of mutants affected
specifically in seed dormancy, which probably rep-
resent genes that control one of the downstream
processes affected by ABA.
phytochrome-B-deficient hy3 mutant suggested to
several authors that screening for insensitivity to FR
might yield phytochrome-A-deficient mutants, as
indeed was later proven. Since phyA mutants have
no obvious phenotype in white light, this specific
screen was required to find them. From the moment
these well defined mutants were available they have
been used to specify the modes of action of these
different phytochromes, e.g. in seed germination
(Botto et al., 1995), anthocyanin formation
(Kerckhoffs et al., 1997) and fiowering (Bagnall et
al., 1995).
2. Photoreceptive pigments
The control of growth and development by the
quality, quantity and duration of light is described as
photomorphogenesis. Plants perceive information
from light through pigment systems such as phyto-
chrome and cryptochrome. The complexity of the
regulation of photomorphogenesis by phytochrome
comes from the fact that different types of phyto-
chrome encoded by at least 45 different genes exist
(Pratt, 1995). These phytochromes differ in their
photo-stability and their temporal and develop-
mental expression. For some processes these dif-
ferent types of phytochrome might have different
modes of action. Mutants at the hyl-hy5 loci, which
are defective in specific aspects of photomorpho-
genesis and which are recognized by their elongated
hypocotyls in white light, were first described by
Koornneef, Rolff & Spruit (1980). Subsequently, the
molecular nature of all five mutants was elucidated
(Table 2). The identification of the blue light
receptor depended fully on the cloning of the HY4
gene (Ahmad & Cashmore, 1993), and the sub-
sequent characterization of the cloned gene (Lin et
al., 1995). The comparison of the phytochrome
chromophore mutants hyl and hy2, which are blind
both to Red (R) and Far Red (FR) light, with the
3. Floral initiation
The transition from the vegetative to the repro-
ductive meristem which produces fiowers, is poorly
understood at the molecular level. To increase our
understanding of this important process in higher
plants a genetic approach has been suggested.
Genetic variation for this trait is abundant. For
instance, dozens of mutants have been found in
Arabidopsis that either delay or advance the tran-
sition to flowering (Haughn, Schultz & Martinez-
Zapater, 1995 ; Peeters & Koornneef 1996), as well as
in other species, but in none of these mutants has
flowering been completely abolished. In contrast to
mutants without a reproductive phase, emf mutants
(Sung et al., 1992) lacking the vegetative phase have
been isolated. The hypothesis that fiowering is the
default state in Arabidopsis, which is repressed by the
EMF products, was then established (Sung et al.,
1992; Martinez-Zapater et al., 1994; Weigel, 1995).
The effect of these product(s) can be modified by
various processes controlled by the flowering-time
genes and also by environmental factors such as light
and temperature. A number of the flowering time
genes, including LD (Lee et al., 1994a), CO
(Putterill et al., 1995) and FCA (Macknight et al,
1997) have now been cloned, and are shown to
Genetics in plant physiology
represent transcription factors {GO and LD) and a
RNA-binding protein {FGA). Using a system with
which the GO gene product is switched on, a careful
analysis of the floral initiation process in relation to
other genes induced by the developmental switch
could be performed (Simon, Igeno &Coupland,
1996). The fact that the genetics of the floral
initiation process indicates a complex regulation
shows that, without genetic dissection, this com-
plexity is hardly accessible for experimental analysis.
THE LARGELY UNEXPLOITED SOURCE,
NATURAL GENETIC VARIATION
The gene pool of a species present in nature contains
allelic variation at many different genes. In contrast
to induced mutants, one does not expect variants
that are strongly affected in vigour, since selection
would have eliminated such genotypes. This limits
this source of variation for basic research, although
this natural variation is the type that has been, and
still is, exploited for plant breeding. Furthermore,
the process of natural selection has led to genotypes
adapted to specific environments. This adaptation
probably reflects variation in ecological/
physiological traits, although morphology often also
differs within the species, which is very obvious in
collections of cultivated plants. This source of
variation, especially that related to physiological
traits, has not been very accessible for genetic
analysis and even less so for molecular genetic
analysis because such properties behave genetically
as quantitative traits, determined by quantitative
trait loci (QTLs), implying polygenic inheritance
and environmental effects on the expression of the
trait. A number of developments in genetics, such as
the progress in marker technology (Rafalski &
Tingey, 1993) and in the improvement of statistical
approaches (Jansen, 1996), made these traits more
accessible and allows the genetic detection of single
loci that determine the genetic variation for such
traits. When single locus differences are identified,
the further molecular analysis might be similar to
that followed wdth monogenic mutants.
The analysis of QTLs is based on the association
of phenotypic differences for the trait of interest with
genetic markers located at specific positions on the
chromosomes, establishing the map position of the
various QTLs. The availability of efficient marker
systems such as microsatellites and AFLPs based on
PCR techniques, enables the molecular analysis in a
far less laborious way as compared with other marker
systems, such as RFLPs and isozymes. The problem
of environmental variation can be solved by using
replications of the individual genotypes. This can be
is achieved by making vegetatively propagated clones
from individual genotypes, especially in outbreeding
species, but also by developing recombinant inbred
lines (RILs, Burr &Burr, 1991) or other homo-
zygous mapping populations such as sets of doubled
haploids (DHs), recombinant backcross lines
(RBLs), also called backcross inbred lines (BILs)
(Ramsey et al, 1996), introgression lines (Us) (Fshed
& Zamir, 1995) or substitution lines. More advanced
material of this kind are near-isogenic lines (NILs),
differing in a small introgression from a corre-
sponding genotype. The multiple use of these
populations without having to genotype the material
again with molecular markers, makes these genetic
stocks extremely valuable. This can be demonstrated
by the analysis of traits as different as flowering
(Jansen et al, 1995) and seed dormancy (van der
Schaar et al, 1997) in the same set of RILs of the two
most widely used Arabidopsis ecotypes, Landsberg
erecta {her) and Columbia (Col). This population
also serves as the standard mapping population in
Arabidopsis (Lister & Dean, 1993). A careful choice
of parents e.g. by choosing extremes of the genot^^pic
variation within a species, extends these oppor-
tunities even more. Examples of' immortal' mapping
populations based on very different genotypes are
the RILs in rice derived from a cross of an upland
japonica variety with an indica lowland variety (Wang
et al, 1994); in barley the DHs have been derived
from crosses between malting and fodder cultivars
(Kleinhofs et al, 1993), and in Arabidopsis crosses
between European and African ecotypes (Alonso-
Blanco et al, unpublished). After the location,
quantification and analysis of the interactions of loci
controlling the trait has been made, it will be
important to characterize the individual loci. In
order to obtain genotypes with only monogenic
differences, the further backcrossing with a recurrent
parent (often one of the parents of the initial cross)
will be necessary, when working with RILs or DHs.
This ' Mendelising' of a QTL can be facilitated by
markers linked to the respective loci and also by
selection of the phenot>-pe in backcross populations.
When NILs are available, this process of
'Mendelising' QTLs has already been performed.
An alternative approach to the dissection of natural
genetic variation is to perform a backcross pro-
gramme with phenotype-based selection from the
beginning (Fig. 1). After a number of backcrosses,
the analysis with molecular markers will indicate
what chromosomal regions of the donor parent are
still present in the selected lines and thereby show
the map position of putative QTLs. Once a NI L
with monogenic segregation has been obtained, the
refinement of the map position can be done in the
progeny of the cross of such a NI L carrying the
introgressed gene, with the recurrent parent. The
selection of recombinants around the locus of
interest, on the basis of recombinants between two
easily scorable outside markers, followed by a
detailed anal3^sis of those recombinants with ad-
ditional markers, will allow the efficient fine-map-
ping necessary for the initiation of map-based
M. Koornneef, C. Alonso-Blanco and A.J. M. Peeters
RP DP
u I I
Parents
FI
o
c
x:
F2
i
3
D
a>
o
o
c
0
o
CO
0
T D
X3
0
0
CO
_ 0
O5
C
F8
I^'L RI L popu lati on
Genotyping with molecular markers covering the genome
Phenotyping for the trait of interest and QTL mapping
1 1
Construction of NI Ls (BILs) containing a single QTL
Physiological and genetical characterisation of the NILs
Figure 1. A schema tic o utline o f the pro ductio n a nd use o f reco mbina nt inbred lines (RILs) a nd nea r iso genic
lmes (NILs). RP, recurrent pa rent; DP, do no r pa rent; S, selected pla nt; BIL, ba ckcro ss inbred line.
clo ning pro cedures. A po tentia l pro blem is tha t it
will be difficult to distinguish if o ne gene o r mo re
tha n o ne very clo sely linked genes determine the
tra its tha t segrega te mo no genica lly. The determi-
na tio n of a very deta iled ma p po sitio n will be
especia lly impo rta nt in tho se species where the
co mplete physica l ma p is a va ila ble a nd fo r which in
the nea r future the co mplete sequence of the geno me
will beco me a va ila ble. When the bio chemica l func-
tio ns of the genes lo ca ted in the regio n of the QTL
a re kno wn, o ne might 'guess' the ca ndida te gene.
Kno wledge of the po sitio n of o pen-rea ding fra mes
will a lso a llo w the selectio n of clo nes tha t ca n be used
fo r tra nsfo rma tio n, which will pro vide the pro o f of
the successful clo ning by co mplementa tio n. Al-
tho ugh in na tura l a lleles it will no t be clea r whether
o ne is dea ling with a lleles tha t ma ke a functio na l
gene pro duct o r with defective a lleles, the deter-
mina tio n of do mina nce ca n give a n indica tio n of this.
To kno ck o ut the wild type a llele, a muta tio na l
a ppro a ch ca n be fo llo wed to find null muta nts. The
deta iled ma p po sitio n is a lso impo rta nt when tra ns-
po so ns a re used fo r this, beca use this will permit the
cho ice of a geno type with a tra nspo sa ble element in
the vicinity of the ta rget gene a nd thereby increa se
the cha nce of finding insertio ns in the ta rget gene,
since tra nspo sa ble elements ha ve a tendency to insert
predo mina ntly to linked sites (Sunda resa n, 1996).
Furthermo re, when ma ny ESTs a re ma pped in the
regio n of interest, it will ena ble the use of DNA
sequences in co mbina tio n with tra nspo so ns to per-
fo rm reverse genetics. Such EST pro bes ca n a lso be
used fo r the detectio n of deletio ns ca used by
irra dia tio n muta genesis.
Exa mples of 'na tura l' mo no genic tra its tha t ha ve
been clo ned a re ma ny disea se resista nce genes (Jo nes
& Jo nes, 1997). Na tura l va ria tio n fo r qua ntita tive
tra its fo r which a lleles a t single lo ci ha ve la rge eflfects
Genetics in plant physiology
and for which map-based cloning efforts have been
initiated are the flowering-time genes FRI (Clarke &
Dean, 1993) and FLC(Lee et al, 19946) in
Arabidopsis.
CONCLUDINGREMARKS
The use of genetics has been successfully exploited
to dissect plant developmental processes. In par-
ticular, the combination of genetics with biochem-
istry and molecular biology allows the study of the
gene functions. The interaction between genes can
be studied by the analysis of double mutants.
However, the classical genetic approach is limited in
specific processes for which mutations are lethal or
have no obvious phenotype. The latter might be due
to redundancy or that the genes have no clear
function under most, or all conditions. For these
situations the reverse genetics approach and the use
of 'trapping' procedures open new possibilities.
Furthermore, the identification of genes with rela-
tively small and general effects will be important
because they might indicate the function of genes
that are overlooked in many mutant isolation experi-
ments. Since this type of genetic variation is more
difficult to analyse, more sophisticated and sensitive
mutants screening techniques need to be developed,
in close collaboration with physiologists and bio-
chemists. The exploitation of natural variation might
be especially fruitful for this, as well as for
understanding how genes are mutated in nature in
order to provide the ecophysiological variation that
gives plasticity to plant species.
ACKNOWLEDGEMENTS
This research was supported by the BIOTECH4 (BIO4-
CT96-0062) and TDRprogramme (BIO4-CT96-5008) of
the European Union.
REFERENCES
Aarts, MGM, Corzaan P, Stiekema WJ , Pereira A. 1995. A
two-element Enhancer-Inhibitor transposon system in Arabi-
dopsis thaliana. Molecular & General Genetics 247: 555-564.
Ahmad M, Cashmore AR. 1993. HY4gene of A. thaliana
encodes a protein with characteristics of a blue-light photo-
receptor. Nature 366: 162-166
Bagnail DJ, King RW, Whitelam GC, Boylan MT, Wagner D,
Quail PH. 1995. Flowering responses to altered expression of
phytochrome in mutants and transgenic lines of Arabidopsis
thaliana (L.) Heynh. Plant Physiology 108: 1495-1503.
Bancroft I. Bhatt AM, Sjodin C, Scofield S, Jones JDG, Dean
C. 1992. Development of an efficient two element tagging
system in Arabidopsis thaliana. Molecular and General Genetics
233: 449-461.
Benning G, Ehrler T, Meyer K, Leube M, Rodriguez P, Grill
E. 1996. Genetic analysis of ABA-mediated control of plant
growth. Abstract in Abscisic acid signal transduction in plants.
Madrid: Juan March Foundation.
Bevan M, Ecker J, Theologis S, Federspeil N, Davis R.
McCombie D, Martiensen R, Chen E, Waterson B, Wilson
R, Rounsley S, Venter C, Tabata S, Salanoubat M, Quetier
F, Cherry J. M., Meinke D. 1997.Objectives: the complete
sequence of a plant genome. Plant Cell 9: 476-478.
Botto JF, Sanchez RA, Whitelam GC, Casals JJ. 1995.
Phytochrome Amediates the promotion of seed germination by
very low fiuences of light and canopy shade light in Arabidopsis
Plant Physiology 110: 439-444.
Burr B, Burr FA. 1991. Recombinant inbreds for molecular
mapping in maize: theoretical and practical consideration
Trends in Genetics 7: 55-60.
Biischges R, Hollricher K, Panstruga R, Simons G, Wolter
M, Frijters A, van Daelen R, van der Lee T, Diergaarde P
Groenendijk J, Topsch S, Vos P, Salamini F, Schulze-
Lefert P. 1997. The barley Mlo gene; a novel control element
of plant pathogen resistance. Cell 88- 695-705
Clarke JH, Dean C. 1993. Mappmg FRI, a locus controlling
flowermg time and vernalization response in Arabidopsis
thaliana . Molecular and General Genetics 242: 81-89.
Cutler S, Ghassemian M, Bonetta D, Cooney S, McCourt P.
1996. Aprotein farnesyl transferase involved in abscisic acid
signal transduction in Arabidopsis. Science 273; 1239-1241
Duckham SC, Linforth RST, Taylor IB. 1991. Abscisic acid
deficient mutants at the aba locus of Arabidopsis thaliana are
impaired in the epoxydation of zeaxanthin. Plant, Cell and
Environment 14: 631-636.
Eshed Y, Zamir D. 1995. An introgression line population of the
cultivated tomato enables the identification and fine mapping of
yield-associated QTL. Genetics 141: 1147-1162.
Feldmann KA. 1991. T-DNA insertion mutagenesis in Arabi-
dopsis: mutational spectrum. Plant Journal 1:71-82.
Finkelstein RR. 1994. Mutations at two new Arabidopsis ABA
responsive loci are similar to abiS mutations. Plant Journal 5-
765-771.
Garner WW, Allard HA. 1920. Effect of relative length of day
and night and other factors of the environment on growth and
reproduction in plants. Journal Agricultural Research 18-
553-606.
Giraudat J, Hauge BM, Valon C, Smalle J, Parcy F,
Goodman HM. 1992. Isolation of the Arabidopsis ABBgene
by positional cloning. Plant Cell 4: 1251-1261.
Giraudat J, Parcy F, Bertauche N, Gosti F, Leung J, Moris P-
C, Bouvier-Durand M, Vartanian N. 1994. Current advances
in abscisic acid action and signalling. Plant Molecular Biology
26: 1557-1577.
Haughn GW, Schultz EA, Martinez-Zapater JM. 1995. The
regulation of fiowering in Arabidopsis thaliayia: meristems,
morphogenesis, and mutants. Canadian Journal of Botanv 73
959-981. ' '
Jansen RC. 1996. Complex plant traits: time for poh^genic
analysis. Trends Plant Science 1: 89-94.
Jansen RC, Van Ooijen JW. Stam P, Lister C, Dean C. 1995.
Genotjrpe by environment interaction in genetic mapping of
multiple quantitative trait loci. Theoretical 8i Applied Genetics
91:33-37.
Jones DA, Jones JDG. 1997. The role of leucine-rich repeat
proteins in plant defences. Advances in Botanical Research,
Advances in Plant Pathology 24: 89-167.
Karssen CM, Brinkhorst-van der Swan DLC, Breekland AE,
Koornneef M. 1983. Induction of dormancy during seed
development by endogenous abscisic acid: studies with abscisic
acid deficient genotypes of Arabidopsis thaliana (L.) Heynh.
Planta 157: 158-165.
Kauschmann A, Jessop A, Koncz C, Szekeres M, Willmitzer
L, Altmann T. 1996. Genetic evidence for the essential role of
brassinosteroids in plant development. Plant Journal 9:
701-713.
Kerckhoffs LHJ, Schreuder MEL, van Tuinen A, Koornneef
M, Kendrick RE. 1997. Phytochrome control of anthocyanin
biosynthesis in tomato seedlings: anah'sis using photomo-
rphogenic plants Photochemistry arid Photobiology 65, 374381.
Kleinhofs A, Kilian A, Saghai Maroof MA, Biyashev RM,
Hayes P, Chen FQ, Lapitan N, Fenwich A, Blake TK,
Kanazin V, Ananiev E, Dahleen L, Kudrna D, Bollinger J,
Knapp SJ, Liu B, Sorrells M, Heun M, Franckowiak JD,
Hoffman D, Skadsen R, Steffenson BJ. 1993. Amolecular,
isozyme and morphological map of the barley {Hordeum vulgare)
genome. Theoretical and Applied Genetics 86: 705-712.
Koes, R. Souer E, van Houwelingen A, Mur L, Spelt C,
Quattrocchio F.,Wing J, Oppedijk B, Ahmed S, Maes T,
Gerats T, Hoogeveen P, Meesters M, Kloos D, Mol JNM.
1995. Targeted gene inactivation in petunia by PCR-based
selection of transposon insertion mutants. Proceedings of the
National Academy of Science, USA 92: 8149-8153.
8 M. Koornneef, C. Alonso-Blanco and A.J. M. Peeters
Koornneef M. Rolff E. Spruit CJP. 1980. Genetic control of
light-inhibited hypocotyl elongation in Arabidopsis thaliana
(L.) Heynh. Zeitschrift fiir Pflanzenphysiologie 100: 147-160.
Lee I, Aukerman MJ, Michaels SD, Weaver IM, John MC,
Amasino RM. 1994a. Isolation oiLUMfNIDEPENDENS :
a gene involved in the control of flowering time in Arabidopsis.
Plant Cell 6: 75-83.
Lee I, Michaels SD, Masshardt AS, Amasino RM. 19946.
The late-flowering phenotype of FRfGfDA and mutations in
LUMINfDEPENDENS is suppressed in the Landsberg erecta
strain of Arabidopsis. Plant Journal 6: 903-909.
Leon-Kloosterziel KM, Alvarez-Gil M, Ruijs GJ, Jacobsen
SE, Olszewski NE, Schvs^artz SH, Zeevaart JAD, Koor-
nneef M. 1996 e. Isolation and characterization of abscisic acid
-deficient Arabidopsis mutants at two new loci. Plant Journal
10: 655-661.
Leon-Kloosterziel KM, van de Bunt GA, Zeevaart JAD,
Koornneef M. 19966. Arabidopsis mutants with a reduced
seed dormancy. Plant Physiology 110: 233-240.
Leung J, Bouvier-Durand M, Morris P-C, Guerrier D,
Chefdor F, Giraudat J. 1994. Arabidopsis ABA-response gene
ABfl: features of a calcium-modulated protein phosphatase.
Science 264: 1448-1452.
Leung J, Merlot S, and Giraudat J. 1997. The Arabidopsis
ABSCISIC ACID-INSENSITIVE 2 (ABI2) and ABIl genes
encode homologous phosphatases 2C involved in abscisic acid
signal transduction. Plant Cell 9: 759-771.
Li J, Nagpal P, Vitart V, McMorris TC, Chory J. 1996. A role
for brassinosteroids in light-dependent development of Arab-
idopsis. Science 111: 398^01.
Lin C, Robertson DE, Ahmad M,Raibekas AA, Schuman
Jorns M, Dutton PL, Cashmore AR. 1995. Association of
flavin adenine dinucleotide with the Arabidopsis blue light
receptor CRYl. Science 269: 968-970.
Lister C, Dean C. 1993. Recombinant inbred lines for mapping
RFLP and phenotypic markers in Arabidopsis thaliana. Plant
Journal . 4: 745-750.
Macknight R, Bancroft I, Page T, Lister C, Schmidt R, Love
K, Westphal L, Murphy G, Sherson S, Cobbet C, Dean C.
1997. FCA, a gene controlling flowering time in Arabidopsis,
encodes a protein containing RNA-binding miotifs and a WW
protein interaction domain. Cell, 89: 737-745.
Marin E, Nussaume L, Quesada A, Gonneau M, Sotta B,
Hugueney P, Frey A, Marion-Poll A. 1996. Molecular
identification of zeaxanthin epoxidase of Nicotiana plumbagini-
folia, a gene involved in abscisic acid biosynthesis and
corresponding to the ABA locus oi Arabidopsis thaliana. EMBO
Journal 15: 2331-2342.
Martinez-Zapater JM, Coupland G, Dean C, Koornneef M.
1994. The transition to flowering in Arabidopsis. In: Meye-
rowitz EM, Somerville CR, eds. Arabidopsis. New York: Cold
Spring Harbor Laboratory Press, 403-434.
Meyer K, Leube MP, Grill E. 1994. A protein phosphatase 2C
involved in ABA signal transduction in Arabidopsis thaliana.
Science 264: 1452-1455.
Oyama T, Shimura Y, Okada K. 1996. HY5 gene: a signal
regulator of photomorphogenesis and root development.
Abstract S65. 7th International Conference on Arabidopsis
Research, Norwich, UK.
Parcy F, Giraudat J. 1997. Interactions between the ABIl and
the ectopically expressed ABI3 genes in controlling abscisic
acid responses in Arabidopsis vegetative tissues. Plant Journal
11: 693-702.
Parks BM, Quail PH. 1991. Phytochrome-deficient hyl and hy2
long hypocotyl mutants of Arabidopsis are defective in phyt-
ochrome chromophore biosynthesis. Plant Cell 3: 1177-1186.
Peeters AJM, Koornneef M. 1996. Genetic variation in
flowering time in Arabidopsis thaliana. Seminars in Cell and
Developmental Biology 7, 381-389.
Pratt LH. 1995. Phytochromes: differential properties, expression
patterns and molecular evolution. Photochemistry and Photo-
biology 61: 10-21.
Putterill J, Robson F, Lee K, Simon R, Coupland G. 1995.
The CON STAN S gene oi Arabidopsis promotes flowering and
encodes a protein showing similarities with a zinc flnger
transcription factor. Cell 80: 847-857.
Rafalski JA, Tingey SV. 1993. Genetic diagnostics in plant
breeding: RAPDs, microsatellites and machines. Trends in
Genetics 9: 275-280.
Ramsay LD, Jennings DE, Bohuon EJR, Arthur AE, Lydiate
DJ, Kearsey MJ, Marshall DF. 1996. The construction of a
substitution library of recombinant backcross lines in Brassica
oleracea for the precision mapping of quantitative trait loci.
Genome 29: 558-567.
Reed JW, Nagpal P, Poole DS, Furuya M, Chory J. 1993.
Mutants in the gene for the red/far-red light receptor
phytochrome B alter cell elongation and physiological responses
throughout Arabidopsis development. Plant Cell 5: 147-157.
Rock CD, Zeevaart JAD. 1991. The aba mutant of Arabidopsis
thaliana is impaired in epoxy-carotenoid biosynthesis. Proceed-
ings of the National Academy of Science, USA. 88:
7496-7499.
Scheres B, Wolkenfelt H, Willemsen V, Terlouw M, Lawson
E, Dean C, Weisbeek P. 1994. Embryonic origin of the
Arabidopsis primary root and root meristem initials. Deve-
lopment 120: 2475-2487.
Schmidt R, West J, Love K, Lenehan Z, Lister C, Thompson
H, Bouchez D, Dean C. 1995. Physical map and organisation
oi Arabidopsis thaliana chromosome 4. Science 270: 480^83.
Schwartz SH, Leon-Kloosterziel KM, Koornneef M, Zeev-
aart JAD. 1997. Biochemical characterisation of the aba2 and
aba3 mutants in Arabidopsis thaliana. Plant Physiology 114:
161-166.
Simon R, Igeno MI, Coupland G. 1996. Activation of floral
meristem identity genes in Arabidopsis. Nature 384: 59-62.
Sommer H, Beltran JP, Huijser P, Pape H, Lonnig WE,
Saedler H, Schwartz-Sommer Z. 1990. Deficiens, a homeotic
gene involved in the control of flower morphogenesis in
Antirrhinum mafus: The protein shows homology to tran-
scription factors. EMBO Journal 9: 605-613.
Sun T-P, Goodman HM, Ausubel F. 1992. Cloning of the
Arabidopsis GAl locus by genomic subtraction. Plant Cell 4:
119-128.
Sundaresan V. 1996. Horizontal spread of transposon muta-
genesis: new uses for old elements. Trends in Plant Science 1:
184-190.
Sundaresan V, Springer P, Volpe T. Haward S, Jones JDG,
Dean C, Ma H, Martienssen R. 1995. Patterns of gene action
in plant development revealed by enhancer trap and gene trap
transposable elements. Genes and Development 9: 1797-1810.
Sung ZR, Belachew A, Shunong B, Bertrand-Garcia R. 1992.
EMF, an Arabidopsis gene required for vegetative shoot
development. Science 25^: 1645-1647.
Tanksley SD, Ganal MW, Martin GB. 1995. Chromosome
landing: a paradigm for map-based cloning in plants with large
genomes. Trends in Genetics 11: 63-68.
Terry M. 1991. Phytochrome chromophore-deflcient mutants.
Plant, Cell and Environment 20: 740-745.
van der Schaar W, Alonso-Blanco C, Leon-Kloosterziel,
Jansen RC, van Ooijen JW, Koornneef M. 1997. QTL
analysis of seed dormancy in Arabidopsis using recombinant
inbred lines and MQM mapping. Heredity 79: 190-200.
Wang G, Mackill DJ, Bonman JM, McCouch SR, Champoux
MC, Nelson RJ. 1994. RFLP mapping of genes conferring
complete and partial resistance to blast in a durably resistant
rice cultivar. Genetics 136: 1421-1434.
Weigel D. 1995. The genetics of flower development: from floral
induction to ovule morphogenesis. Annual Review of Genetics
29: 19-39.
Whitelam G, Johnson E, Peng J, Carol P, Anderson ML,
Cowl JS, Harberd NP. 1993. Phytochrome A null mutants of
Arabidopsis display a wild-type phenotype in white light. Plant
Cell 5: 757-768.
Zachgo EA, Wang ML, Dewdney J, Bouchez D, Camilleri C,
Belmonte S, Huang L, Dolan M, Goodman HM. 1996. A
physical map of chromosome 2 oi Arabidopsis thaliana. Genome
Research 6: 19-25.