Maize Genetics Cooperation Newsletter vol 81 2007
In
maize, the zein synthesizing system is particularly adapted for the study of
the regulating mechanisms of plant genes because i) its expression is
restricted to specific tissues and stages of seed development and ii) because
of the availability of mutants useful in dissecting the regulatory processes
taking place in the developing seed (Pirona et al., Maydica 50:515-530,
2005). Studies on genetic
mutations that affect the accumulation of different zeins have demonstrated the
existence of several regulatory signals controlling the expression of specific
members of the zein family which confer an opaque phenotype to the endosperm
(Motto et al., pp: 479-522, In: B. A. Larkins and I. K. Vasil (eds.), Cellular
and Molecular Biology of Plant Seed Development, Kluwer Acad. Publ., The
Netherlands, 1997). For example,
the recessive mutations opaque2 (o2) and
opaque7 (o7) induce specific decreases in accumulation of 22 and 19-kD
alpha-zeins, respectively, while the opaque15
(o15) mutation exerts its effect primarily on the 27-kD gamma zeins. The recessive mutation opaque6 (o6) and the dominant or
semi-dominant mutations Floury (Fl2),
Defective endosperm *B30 (De*B30),
and Mucronate (Mc) cause a more
general reduction in accumulation of all zein classes.
The
o2 mutation has been widely studied
at the molecular, genetic, and biochemical levels (see Pirona et al.,
2005). The product of the o2 gene is a basic leucine zipper (bZiP)
transcriptional regulator that is specifically expressed in the endosperm and
activates the expression of 22 kDa alpha-zein and 15 kDa gamma-zein, together
with the B-32 gene, encoding an
endosperm specific ribosome nactivating protein. Other possible direct or indirect target genes of the o2 factor have been shown to belong to
various metabolic pathways, suggesting that o2
may play an important role in the developing grain, as a coordinator of the expression
of storage protein, and nitrogen and carbon metabolism genes.
In
recent years, the development of extensive maize cDNA libraries, along with
computer software to systematically characterize them, has made it possible to
analyze gene expression in developing maize endosperm more thoroughly. Accordingly, we have used cDNA
microarray technology to investigate the transcription profiles and
differential gene expression of maize endosperm from two different opaque
mutants (o2 and o7) and in double mutant combination (o2o7).
Microarrays
were assembled using clones obtained from the EC ZeaStar project ( Edwards et
al., unpublished results).
Briefly, 20 part-normalized cDNA libraries were prepared from tissues
covering 5 key stages in both endosperm and kernel development. Approximately 20,000 ESTs were
sequenced, aligned, assembled into contigs using a similarity score of 80%, and
annotated using BLASTA and TBLASTN software. Contigs and singleton cDNAs were used to construct a unigene
set of 8,950 sequences. EST
sequences were analyzed with the BLAST2GO software
(http://www.blast2go.de). First,
homology searches using public domain non-redundant databases were performed
and identified significantly homologous sequences for 48.4% of the ESTs considered. These ESTs represented 3,090 single hit
(71.3%) and 1,240 multiple hit sequences.
Subsequently, an attempt was made to associate biological functions to
each of the ESTs showing sequence homology using the gene ontology
(http://www.geneontology.org) and KEGG databases (http://www.genome.jp/kegg). Approximately 85% of the ESTs analyzed
could be associated with GO database entries. The results of this analysis permitted us to divide the
aforementioned ESTs into 24 functional groups with a total of 7,250 clones identified as duplicates.
Microarray
slides containing the entire Zeastar unigene set were hybridized with probes
derived from endosperm tissue harvested 15 days after pollination (DAP) and
derived from the A69Ywt, A69Yo2, A69Yo7, and A69Yo2o7 isogenic
lines. To reduce hybridization
artifacts, all probes were labelled both with Cy3 and with Cy5 and used in
dye-swapping experiments on a series of three independent slides. The expression data obtained were
assayed for consistency by performing T-tests at 95% confidence levels.
All
microarray experiments were performed in triplicate using dye swapping, hence
giving rise to 12 independent measurements for each EST, considering the
presence of duplicate spots on each slide. Raw measurements of spot fluorescence intensities were
collected from hybridized slides using a Genepix 4100A scanner and Genepix Pro4
software (Axon Instruments, Union, CA).
Subsequently, the spot values obtained were corrected for background
fluorescence and analyzed using the Vector Xpression3 software (Informax,
Frederick, MD). The data were log2
transformed and normalized by equalizing the mean intensity of each channel to
1. To verify reproducibility
between spots and between channels, T tests were performed applying a 95%
confidence threshold and allowing us to remove inconsistent hybridization
results. Ratios between wild type
and mutant expression levels were calculated and ESTs exhibiting ratios below
0.5 or over 2 were selected for further analysis.
Average
signal values derived from the four probes used were graphed using a
logarithmic scale. The graphical
representations clearly showed the prevalence of genes with distinct expression
patterns in the A69Ywt and A69Yo2 genotypes. Conversely, the A69Ywt
and A69Yo7 genotypes show less
evident differences in expression levels.
The A69Yo2o7 double mutant
exhibits differences in expression patterns resembling those obtained for the
A69Yo2 genotype. A plot of A69Yo2 vs. A69Yo7 expression
levels showed the cumulative effect of both genotypes, revealing a high number
of genes with distinct expression patterns.
Consistently
performing spots in T-tests were selected and used to calculate wtl mutant expression ratios. Among the ESTs considered, 17.1%
exhibited a down-regulated expression profile. The o2 mutation
may be associated with 649 down-regulated ESTs. 508 down-regulated ESTs were identified in the A69Yo7 background, whereas 759 ESTs showed a
reduced expression pattern in A69Yo2o7. Up-regulated expression profiles
were found for 3.23% of the ESTs considered. One hundred and thirteen up-regulated ESTs were identified
in the A69Yo2, 26 in the A69Yo7, and 86 in an the A69Yo2o7 backgrounds, respectively. Among the ESTs identified, 36.7%
exhibited relevant homology with sequences deposited in public databases and
could be univocally associated with known biological processes related to amino
acid and carbohydrate metabolism, signal transduction, protein turnover,
transport and protein folding. In
addition, three transcription factors other than o2 appear to be down-regulated. Collectively, the results may provide a framework for
investigating a common mechanism that underlies the o2 and o7 kernel
phenotypes.
Please Note: Notes
submitted to the Maize Genetics Cooperation Newsletter may be cited only with
consent of authors.