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Microarrays and Expressed Sequence Tags (ESTs) for Developing
Seeds of Arabidopsis |
Comparatively little research has
focused on the molecular biology of Arabidopsis seeds because of
technical difficulties associated with their small size. This is
regrettable because seeds, with their remarkable abilities to store
food, become dehydrated, and attain dormancy, are fascinating
biological structures. Such a dearth of knowledge is equally
unfortunate from an applied perspective, given that Arabidopsis would
be an especially good model system for understanding the molecular
biology of closely related oilseed crops such as rape (Brassica
napa). In this issue, Girke et al. (pp. 1570-1581)
report on their successful production of microarrays that display
approximately 2,600 of the genes that are expressed in developing
Arabidopsis seeds (Fig. 1). The DNA for the genes spotted on the arrays
were selected from more than 10,000 clones that were partially
sequenced from a cDNA library of ESTs. Approximately 25% of these
2,600 genes are expressed at ratios that are 
2-fold greater
than in leaves or roots, whereas about 10% are expressed at ratios
that are 10-fold greater. Included in this list are a large
number of proteins of unknown function, and potential regulatory
factors such as protein kinases, phosphatases, and transcription
factors. In a companion paper, White et al. (pp. 1582-1594)
measure the relative levels of these 2,600 ESTs as a method for
exploring the primary metabolic routes for the conversion of
photosynthate into oil in developing seeds of Arabidopsis. Much of
their data provides elegant confirmation of conclusions previously
drawn by conventional biochemistry. However, the authors also provide
many fascinating insights into such questions as the relative
importance of cytosolic versus plastidic glycolysis in the conversion
of carbohydrates into precursors of fatty acids, the possible
role of photosystem I in providing reducing equivalents (the seeds are
green early in development), and the nature of the transient
accumulation of starch during seed filling. The authors also note that
the ESTs encoding proteins in a specific section of a given metabolic
pathway tend to show similar abundance, perhaps indicating the
existence of metabolic regulons or groups of genes that are
coordinately expressed. Such regulons would be obvious targets in the
modification of seed storage compositions by genetic
engineering.
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Figure 1.
. Microarray segments (in false
color presentation) depict differential expression of leaf (green) and
seed (red) ESTs.
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Cytokinin-Activated Genes |
Cytokinins affect the expression of many genes in
plants, but most of these genes are regulated additionally by other
stimuli or are induced after a long lag phase or both. Exceptions
include the ARR4 and ARR5 genes that display
properties more in keeping with them being primary response
genes. The heightened transcription of these two genes in
response to cytokinin is rapid, specific, and resistant to inhibitors
of protein synthesis. Moreover, their DNA sequences, which are similar
to those of bacterial two-component response regulators, also indicate
that ARR4 and ARR5 may play a role in the early
steps of cytokinin signal transduction. In this issue,
D'Agostino et al. (pp. 1706-1717) report on their
discovery of several more ARR-type genes in Arabidopsis, and
shed light on the mechanism of enhancement of ARR5
transcript levels by cytokinin and the pattern of its expression in
Arabidopsis. In theory, an increase in steady-state levels of
ARR5 mRNA in response to cytokinin could be due to
increased transcription or the stabilization of the existing
message or both. By incorporating a 
-glucuronidase reporter gene
downstream of the ARR5 promoter and performing a nuclear
run-on assay, the authors were able to show that the accumulation
of ARR5 transcript was due partially, perhaps wholly, to
enhanced transcription. This same reporter construct and whole mount in
situ analyses were used to probe the pattern of expression of
ARR5. The highest levels of expression were observed in the
root and shoot apical meristems, at the junction of the pedicle and
fruit, and the central portion of mature roots (Fig.
2).
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Figure 2.
A -glucuronidase reporter reveals the intense
transcription of the cytokinin-induced gene ARR5 in Arabidopsis root
apical meristem.
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G-Protein Regulators |
Two major classes of G proteins are involved in cell
signaling in eukaryotic cells: heterotrimeric G proteins and the Ras superfamily of small monomeric GTPases. Of the five major
families of monomeric G proteins that compose the Ras superfamily, only the RHO family (termed "Rops" by plant biologists) is known to be
well-represented in plants. Mutant studies have revealed that Rops play
a pivotal role in a diversity of processes in plants, including
tip growth, the development of polarity, cell morphogenesis, cell wall
synthesis, hydrogen peroxide production, programmed cell death, and
probably hormone responses. In animals and yeasts, indispensible
proteins (Rho GTPase-activating proteins [GAPs]) control RHO
GTPase activity. This has caused plant biologists to wonder
whether similar activating proteins also control the activity of Rops.
Complementing a recent report of Rho GAP-like proteins in lotus,
Wu et al. (pp. 1625-1636) identify several similar proteins
in Arabidopsis by means of a yeast two-hybrid assay. These
"RopGAPs" specifically stimulate the hydrolysis of GTP by Rop
GTPases but not Cdc42 GTPases. Both the lotus and the Arabidopsis
RopGAPs, unlike animal and fungal GAPs, have a Cdc42/Rac-interactive binding motif in their N-terminal region. By deletion and point mutation experiments, Wu et al. demonstrate that the
Cdc42/Rac-interactive binding domain is involved in the formation or
stabilization of the transitional state of Rop GTPases.
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Mapping Markers Become Single Nucleotide Amplified
Polymorphisms (SNAP) |
Map-based positional cloning has traditionally been a
standard but time-consuming and expensive procedure for the
isolation of genes defined by mutation. The main obstacle encountered
in map-based cloning approaches has been the insufficient number of
PCR-based molecular markers available for fine-structure mapping. In
this issue, Drenkard et al. (pp. 1483-1492) describe a new,
inexpensive, and efficient PCR-based mapping strategy that employs
modified allele-specific primers and standard molecular biological
equipment. The key to this new technique is the utilization of primers
that have tailored mismatches near their 3' end that allow for the
preferential amplification of one allele relative to another. The
addition of an extra mismatch to the primer, in addition to the
presence of the natural mismatch at the 3' end, produces a dramatic
reduction in PCR product yield of the nonspecific allele but has only
minor effects on the amplification of the specific allele. The design
of SNAP markers takes advantage of a computer program (SNAPER)
that was written based on a set of empirical data that evaluates
the addition of different mismatch alternatives on PCR amplification.
The SNAP procedure, in conjunction with Cereon Genomics' (Cambridge,
MA) laudable release to the public of approximately 25,000 single nucleotide polymorphisms between the Columbia and
Landsberg erecta accessions, should allow for the
efficient design of mapping markers virtually anywhere in the genome.
As an example of the power of this new technique, the authors report
that they were able to localize the disease resistance mutation
edr5-1 to a 315-kB region on the long arm of chromosome 4 in approximately a 3-week period (excluding the time required for
primer synthesis). Further mapping is in progress.
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Glu Receptors in Plants |
Ionotropic Glu receptors (iGluRs) are the predominant
neuroreceptors in the mammalian brain. There, they mediate rapid
chemical transmission across synapses by increasing the permeability of the post-synaptic membranes to K+, Na+, and
Ca2+ ions. The influx of Ca2+ ions, in
particular, is associated with the occurrence of long-term potentiation
an important neurophysiological process underlying memory
and learning. Many questions are raised, therefore, by the recent
discovery that this month's honored organism, the brainless and
slow-witted Arabidopsis, has genes that are homologous to those that
code for iGluRs in animals. Do these Arabidopsis genes, in fact, code
for functional iGluR-like receptors and, if so, then in what cells and
membranes do they occur? More fundamentally, what function do these
putative Glu receptors serve? In this issue, Brenner et al. (pp.
1615-1624) examine the effects of a cycad-derived iGluR agonist
on the growth and morphology of Arabidopsis. Light-grown seedlings
treated with this drug showed a 2-fold increase in hypocotyl
elongation, an effect that was reversible by the simultaneous
application of Glu (presumably the natural agonist of this receptor).
Three classes of mutants were isolated that showed normal growth
in the light in the presence of the iGluR agonist. One class shows
abnormal growth in the dark even in the absence of the drug, suggesting
that plant Glu receptors may play a role in skotomorphogenesis. In a
related paper, Dennison and Spalding (pp. 1511-1514) report
that Glu causes a rapid increase in
[Ca2+]cyt in transgenic
Arabidopsis expressing the Ca2+-sensitive
photoprotein aequorin. This effect, which is accompanied by a
depolarization of the membrane potential, is not induced by other amino
acids, and is blocked by La3+ (a nonspecific
Ca2+ channel blocker).
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Microarrays and Expressed...
Cytokinin-Activated Genes