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.2008 Apr 21:8:43.
doi: 10.1186/1471-2229-8-43.

A spatial dissection of the Arabidopsis floral transcriptome by MPSS

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A spatial dissection of the Arabidopsis floral transcriptome by MPSS

Jason A Peiffer et al. BMC Plant Biol..

Abstract

Background: We have further characterized floral organ-localized gene expression in the inflorescence of Arabidopsis thaliana by comparison of massively parallel signature sequencing (MPSS) data. Six libraries of RNA sequence tags from immature inflorescence tissues were constructed and matched to their respective loci in the annotated Arabidopsis genome. These signature libraries survey the floral transcriptome of wild-type tissue as well as the floral homeotic mutants, apetala1, apetala3, agamous, a superman/apetala1 double mutant, and differentiated ovules dissected from the gynoecia of wild-type inflorescences. Comparing and contrasting these MPSS floral expression libraries enabled demarcation of transcripts enriched in the petals, stamens, stigma-style, gynoecia, and those with predicted enrichment within the sepal/sepal-petals, petal-stamens, or gynoecia-stamens.

Results: By comparison of expression libraries, a total of 572 genes were found to have organ-enriched expression within the inflorescence. The bulk of characterized organ-enriched transcript diversity was noted in the gynoecia and stamens, whereas fewer genes demonstrated sepal or petal-localized expression. Validation of the computational analyses was performed by comparison with previously published expression data, in situ hybridizations, promoter-reporter fusions, and reverse transcription PCR. A number of well-characterized genes were accurately delineated within our system of transcript filtration. Moreover, empirical validations confirm MPSS predictions for several genes with previously uncharacterized expression patterns.

Conclusion: This extensive MPSS analysis confirms and supplements prior microarray floral expression studies and illustrates the utility of sequence survey-based expression analysis in functional genomics. Spatial floral expression data accrued by MPSS and similar methods will be advantageous in the elucidation of more comprehensive genetic regulatory networks governing floral development.

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Figures

Figure 1
Figure 1
Transcriptional diversity of floral organs, as predicted by MPSS. The number of genes matching our stringent filtration of MPSS expression libraries for each organ-enriched set of genes.
Figure 2
Figure 2
Expression of genes with putative organ-enriched expression relative to wild type in homeotic mutants with expected over expression. Homeotic mutant expression levels of genes with organ-enriched expression were compared to wild type expression. The proportion of genes over-expressed (p < 0.05), under-expressed (p < 0.05), and not significantly different (p > 0.05) relative to wild type were determined within each organ-enriched gene set and homeotic mutant of expected over expression. Values on bars refer to the number of genes within each data set.
Figure 3
Figure 3
Empirical validation viain situhybridization.(A-C)At1g33430 (anti-sense probe), (D-E) (sense control probe) noted signal on stamen and carpel primordial with strong expression detected in the tapetum and microspore as predicted by MPSS.(F-G)At2g19070 (anti-sense probe), (H)(sense control probe) signal identified on the tapetum. Stamen and petal localized expression predicted by MPSS(I-J)At1g54860 (anti-sense probe) signal on stigmatic papillae as well as septum and developing microspore. Predicted to be stamen-enriched by MPSS analysis.(K-L)At5g59810 (anti-sense probe) transient signal noted on a specific microspore of the tetrad. Carpel-enrichment predicted by MPSS.(M)At1g72290 (anti-sense probe) signal identified on the septum. Predicted to be localized within the stamen and the carpel by MPSS.(N)At2g42940 (anti-sense probe)(O)(sense control probe) weak signal on stamen and carpel primordial. Identified as a putative stamen-enriched transcript by MPSS.(P)AP3 (anti-sense control probe).
Figure 4
Figure 4
Empirical validation by promoter:GUS fusions.(A)Wild type control.(B-E)At1g20130[B], At3g27025[C], At2g42940 ([D]and[E]). MPSS-predicted stamen-enriched promoter:GUS.(F-G)At1g07370[F], At1g27900[G]. MPSS-predicted carpel-enriched promoter:GUS.(H)At1g33430. MPSS-predicted stamen/carpel-enriched promoter:GUS.(I-J)At5g07550[I], At2g19070[J]. MPSS-predicted stamen/petal-enriched promoter:GUS.(K)At1g07930. MPSS-ap3expression enriched relative to other mutants.(L)At2g35340. MPSS-predicted absence of floral expression.(M)At1g26270. MPSS-predicted ubiquitous floral expression.(N)At1g68200. MPSS-ap1sole expression.(O)At2g43100. MPSS-ap3expression enriched relative to other mutants.(P)At3g15160. MPSS predicted absence insup ap1mutant.(Q)At52900. MPSS-ap3andagenriched expression.
Figure 5
Figure 5
Expression patterns conferred by promoter:uidA fusions and reverse transcription PCR corresponding to genes with enriched ovule expression. Promoter:uidAfusions. Absence of GUS expression in additional floral organs (sepals, petals, stamens and gynoecia) was confirmed for all lines except for At2g47470 that shows expression in the carpel walls and stigma (data not shown).(A)At1g05550; GUS is expressed in both integuments and the nucellus, at the chalazal region.(B)At5g24420; GUS is expressed in the funiculus and both integuments throughout the ovule.(C)At5g49180; GUS is expressed in both integuments and the funiculus, but not in the female gametophyte.(D)At3g06240; GUS is expressed in the anatropus integumentary ridge and a small region of the dorsal outer integument.(E)At1g27330: GUS is expressed in the micropylar pole and both integuments.(F)RT-PCR expression of genes predicted to be specifically expressed in the ovule. Total RNA was extracted from individual floral organs and used for reverse-transcriptase PCR analysis. Lane 1: fully differentiated ovules; Lane 2: petals; Lane 3: sepals. Amplified fragment sizes: At4g04620 (231 bp); At1g19240 (250 bp); At4g27860 (235 bp); At4g14420 (209 bp); At2g42710 (223 bp).
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