Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants
BMC Plant Biology
Expression-based discovery of candidate ovule development regulators through transcriptional profiling of ovule mutants
Debra J Skinner 0 1
Charles S Gasser 1
0 Department of Crop Science, University of Illinois , Urbana, IL 61801 , USA
1 Department of Molecular and Cellular Biology, University of California , Davis, CA 95616 , USA
Background: Arabidopsis ovules comprise four morphologically distinct parts: the nucellus, which contains the embryo sac, two integuments that become the seed coat, and the funiculus that anchors the ovule within the carpel. Analysis of developmental mutants has shown that ovule morphogenesis relies on tightly regulated genetic interactions that can serve as a model for developmental regulation. Redundancy, pleiotropic effects and subtle phenotypes may preclude identification of mutants affecting some processes in screens for phenotypic changes. Expressionbased gene discovery can be used access such obscured genes. Results: Affymetrix microarrays were used for expression-based gene discovery to identify sets of genes expressed in either or both integuments. The genes were identified by comparison of pistil mRNA from wild type with mRNA from two mutants; inner no outer (ino, which lacks the outer integument), and aintegumenta (ant, which lacks both integuments). Pools of pistils representing early and late stages of ovule development were evaluated and data from the three genotypes were used to designate genes that were predominantly expressed in the integuments using pair-wise and cluster analyses. Approximately two hundred genes were found to have a high probability of preferential expression in these structures, and the predictive nature of the expression classes was confirmed with reverse transcriptase polymerase chain reaction and in situ hybridization. Conclusion: The results showed that it was possible to use a mutant, ant, with broad effects on plant phenotype to identify genes expressed specifically in ovules, when coupled with predictions from known gene expression patterns, or in combination with a more specific mutant, ino. Robust microarray averaging (RMA) analysis of array data provided the most reliable comparisons, especially for weakly expressed genes. The studies yielded an over-abundance of transcriptional regulators in the identified genes, and these form a set of candidate genes for evaluation of roles in ovule development using reverse genetics.
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Background
Ovules, the precursors to seeds, are an important focus of
study to better understand plant development within a
unique reproductive context. Ovules are highly
specialized for reproductive function, but the typical angiosperm
ovule, as found in Arabidopsis, is relatively simple
morphologically. Development of the ovule within the carpel
is well described, [1-5], beginning with primordia
emergence from the marginal placentas of the carpels (floral
stage 9, ovule stage 1). The primordia have three regions,
the distal region or nucellus, marked by the formation of
the large megaspore mother cell, the central or chalaza
region indicated by the emergence of the two
integuments, and the proximal region which forms the funiculus
supporting the ovule (Figure 1A; floral stage 10, ovule
early stage 2). The inner integument initiates as a ring
from divisions in the L1, while the outer integument
derives from divisions on the gynobasal side of the ovule
below the inner integument. The integuments grow
together to enclose the nucellus and when this has
occurred the embryo sac develops from a meiotic product
of the megasporocyte. The integuments continue to
differentiate with the outer and inner integument cells
changing in appearance in preparation for the integuments roles
FOivguulereph1enotypes of wild type, ino and ant
Ovule phenotypes of wild type, ino and ant. A
comparison of wild type (A C) ovule development with ino (D F)
and ant (G I) using scanning electron and fluorescence
microscopy. Ovules are shown at developmental stage 2-IV
(A, D, G), and 4-IV (B, E, H). (A, B) In wild type ovules, the
two integuments grow as sheaths around the nucellus until it
is fully enclosed and the outer integument envelopes the
inner integument. (D, E) In contrast, ino mutant ovules show
only inner integument growth and this structure encloses the
nucellus but does not cause curvature of the ovule at
maturity. (G, H) ant ovules do not initiate integuments but do
elongate and form a swollen region at the chalaza. The ant
nucellus is naked at maturity. (C, F, I) Ovules at anthesis
were cleared and stained for callose accumulation to identify
non-functional embryo sacs that can be seen as brightly
fluorescing structures in ino mutants (arrow, F) and that are
absent in wild type (C). Mature ant ovules (I) lack embryo
sacs, but show callose fluorescence associated with chalaza
and nucellus. c, chalaza; f, funiculus; ii, inner integument; oi,
outer integument; n, nucellus. Scale bar = 15 m in A, D, and
G; 20 m (...truncated)