Annals of Botany 2008 101(7):NP; doi:10.1093/aob/mcn060
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John Bryant takes a closer look at some of this month's Original Articles
J. A. Bryant, Professor
University of Exeter, UK
E-mail j.a.bryant{at}exeter.ac.uk
Lost loci in Primula parentage probe
I have often heard
it said that speciation is a slow process, taking place over
many generations, even when populations are separated. There
is certainly much truth in that statement but equally it ignores
genetic events that create new species more or less instantly.
One of those events is polyploidization. A glance at chromosome
numbers in almost any angiosperm genus reveals that polyploidization
has played a major role in angiosperm evolution. Indeed,
Guggisberg et al. from Zurich, Switzerland (pp. 919–927) cite a review by Soltis (2005. Ancient and recent polyploidy
in angiosperms.
New Phytologist 166: 5–8), which states
that at least 70 % of angiosperms have polyploid origins. Modern
molecular and cytological techniques help investigate putative
parental origins of polyploids as has been used here by the
Zurich group for a North American
Primula species,
P. egaliksensis (2
n = 40). This is believed to be an allopolyploid derived from
a hybrid between the relatively widely diverged
P. mistassinica (2
n = 18) and
P. nutans (2
n = 22). Hybridization of either
P. mistassinica or
P. nutans DNA to chromosome spreads of
P. egaliksensis revealed that both putative parental chromosome sets are present
in
P. egaliksensis, with no evidence for major chromosomal re-arrangements
between the two genomes. However, focus on the genes encoding
the major rRNAs (‘rDNA’) revealed some more local
changes, in particular the loss in the hybrid of the maternal
parent's 45S rDNA sequences. Further, heterochromatic knobs
carrying the rDNA internal transcribed spacer (ITS), present
in the maternal parent
P. mistassinica, are absent in the hybrid,
while sequencing of 44 clones of the ITS of the hybrid revealed
that only 5 % were of
P. mistassinica origin. The loss of the
maternal rDNA sequences may have resulted from nucleolar dominance
while the more general lack of chromosome re-arrangement and
genome homogenization may be a result of the taxonomic distance
between the two parents.
Roles of rols in root regulation
Like its close relative
Agrobacterium tumefaciens,
A.rhizogenes is a naturally occurring
agent of plant genetic transformation; it is the causative agent
of ‘hairy root’ disease in which roots proliferate
at the infection site. Two groups of ‘oncogenes’
on the root-inducing plasmid, the
aux genes and the
rol genes,
are important for this transformation. Interestingly, as pointed
out by
Alpizar et al.(Montpellier, France, pp. 929–940),
the roots may be excised from the plant and grown readily in
culture, and used for regeneration of whole plants and the study
of root growth and physiology. In common with
A. tumefaciens,
A.rhizogenes shows differing abilities to infect different dicot
plants. For both bacteria, coffee (
Coffea arabica) is a difficult
host (although methods for generating GM coffee have now been
developed). The authors have previously achieved transformation
of coffee with
A. rhizogenes but the excised roots did not proliferate
and soon died. In the new series of experiments, 62 different
hairy root clones were grown under different conditions. All
of the clones were shown by PCR to have the
rolB and
rolC oncogenes
integrated into their genome but none of them had the bacterial
aux genes. Added auxin was thus required for proliferation of
all clones and was optimally supplied as 0·5 µ
M IBA. Sucrose was not essential but its inclusion at 2 % (w/v)
was optimal for root growth with low (20 µmol m
–2 s
–1) light intensities or darkness favouring regeneration.
Under optimal conditions, the 62 clones have been maintained
and sub-cultured for over 3 years. Although there is some variation
between clones in respect of root length and the proportion
of fine roots, the majority of their phenotypes did not differ
significantly from the roots of non-transformed plants. They
therefore constitute a useful system for study of root characters
such as resistance to nematodes.
ALS well in mutant medic
‘Test the
trait, not the breeding method’ has almost become a mantra
in my own contributions to the UK's debate on GM crops. For
me, this point is well illustrated in the paper by
Oldach et al., Urrbrae, South Australia (pp. 997–1005).
The authors describe the use of sulfonylurea (SU) herbicides
with cereal crops. Although these herbicides are regarded as
safe, nevertheless their rate of degradation in alkaline soils
is slow; slow enough to affect the annual medics (
Medicago spp.)
grown in rotation with the cereals. An SU-tolerant cultivar
of
M. littoralis (‘Angel’) has been developed by
mutagenesis-based breeding and the main aim of the authors has
been to identify the molecular basis of this mutation. Analysis
of segregation ratios of the
F2 populations from crosses between
‘Angel’ and intolerant
M. trunculata indicated a
single dominant gene. Based on the mode of action of SU herbicides,
this was likely to be a mutant form of
ALS, the gene that encodes
acetolactate synthase, an enzyme involved in synthesis of branched-chain
amino acids. The sequence of the
Arabidopsis thaliana ALS gene
was used to interrogate the
Medicago database, revealing two
homologues located respectively on chromosomes 2 and 3. Linkage
analysis using known markers then revealed that the herbicide-tolerance
trait was associated with a region of the chromosome 3 containing
the
ALS locus. Sequencing of the wild-type and mutant
ALS genes
revealed a single amino-acid change from proline to leucine.
These data facilitated the development of a diagnostic marker
for SU tolerance while RT–PCR showed that ‘Angel’
does indeed express the mutant
ALS. The way is thus open for
use of the mutant allele in
Medicago breeding programmes. However,
a similar mutant
ALS from
Arabidopsis has been used to transform
Nicotiana and
Brassica napus by GM techniques. To return to
my opening comment: which is more important here, the genetic
trait or the breeding method?
Pump up the volume
Scientific progress
involves both expanding the boundaries of what we know and,
from time to time, revising our ideas about what we thought
we already knew. The paper by
Lechner et al. from Balcarce, Argentina and Montpellier, France (pp. 1007–1015) provides a clear example of the second category. They grew
Arabidopsis thaliana and
Helianthus annuus plants under water deficit and
under well-watered conditions. In both species, water deficit
markedly reduced leaf growth rate, although the period of leaf
expansion was similar in the two treatments. Final leaf area
in the droughted plants was therefore much smaller than that
of control plants. When droughted plants were re-watered, leaves
still in the expansion phase showed a much increased expansion
rate. However, the really surprising result was that leaves
of droughted plants that had stopped expanding (and were assumed
therefore to have reached their ‘final’ size) started
expanding again. This was especially dramatic in
A. thaliana,
with some leaves increasing their area by up to 186 %. Increases
in
H. annuus were smaller (up to 27 %) but nevertheless significant.
The increases in area did not involve cell division but were
solely caused by cell expansion. Analysis of the response in
relation to leaf age showed that the longer the gap between
the cessation of expansion in droughted conditions and its re-initiation
induced by re-watering, the smaller was the response, eventually
declining to zero in the oldest leaves. This indicates that
there is a ‘developmental window’ during which it
is possible for leaf cells to resume expansion growth. Under
the conditions used in these experiments, the window was 4 days
in
H. annuus and 11 days in
A. thaliana. This window is likely
to represent a period in which the biophysical/biochemical changes
in the cell wall are making the wall more rigid but until full
rigidity is achieved, the wall is able to respond to increased
turgor pressure.
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