Abstract
BackgroundNectaries are the floral organs responsible for the synthesis and secretion of nectar. Despite their central roles in pollination biology, very little is understood about the molecular mechanisms underlying nectar production. This project was undertaken to identify genes potentially involved in mediating nectary form and function in Brassica rapa.Methodology and Principal FindingsFour cDNA libraries were created using RNA isolated from the median and lateral nectaries of B. rapa flowers, with one normalized and one non-normalized library being generated from each tissue. Approximately 3,000 clones from each library were randomly sequenced from the 5′ end to generate a total of 11,101 high quality expressed sequence tags (ESTs). Sequence assembly of all ESTs together allowed the identification of 1,453 contigs and 4,403 singleton sequences, with the Basic Localized Alignment Search Tool (BLAST) being used to identify 4,138 presumptive orthologs to Arabidopsis thaliana genes. Several genes differentially expressed between median and lateral nectaries were initially identified based upon the number of BLAST hits represented by independent ESTs, and later confirmed via reverse transcription polymerase chain reaction (RT PCR). RT PCR was also used to verify the expression patterns of eight putative orthologs to known Arabidopsis nectary-enriched genes.Conclusions/SignificanceThis work provided a snapshot of gene expression in actively secreting B. rapa nectaries, and also allowed the identification of differential gene expression between median and lateral nectaries. Moreover, 207 orthologs to known nectary-enriched genes from Arabidopsis were identified through this analysis. The results suggest that genes involved in nectar production are conserved amongst the Brassicaceae, and also supply clones and sequence information that can be used to probe nectary function in B. rapa.
Highlights
Floral nectar is the primary reward offered by angiosperms to attract pollinators [1]
When performing expressed sequence tag (EST) analysis, normalized libraries are generally useful for identifying all genes that may be expressed in a given tissue, including ones expressed at low levels [18], whereas nonnormalized libraries are better suited for providing an indication of gene expression level by the total number of redundant or overlapping expressed sequence tags (ESTs)
ALocus identified by blastx searches of B. rapa contigs against Arabidopsis proteins (TAIR9 annotation release). bNumber of ESTs within contigs and singleton sequences generating hits against Arabidopsis loci, listed by individual library. cAffymetrix ATH1 mean signal probe ratio; original raw data from [17]. dDifferential expression verified by reverse transcription polymerase chain reaction (RT PCR), results shown in Fig. 3. eExpression pattern previously verified for ortholog to A1g77110 (Ruhlmann and Carter, in preparation). doi:10.1371/journal.pone.0008782.t002
Summary
Floral nectar is the primary reward offered by angiosperms to attract pollinators [1]. Median nectaries occur on opposite sides of the flower but only in between the insertion points of two long stamen [7]. Both lateral and median nectaries are well-differentiated organs and are subtended by phloem, which is thought to provide most ‘‘pre-nectar’’ components (e.g., sucrose, amino acids, etc.) to the nectary [5,9]. Nectaries are the floral organs responsible for the synthesis and secretion of nectar Despite their central roles in pollination biology, very little is understood about the molecular mechanisms underlying nectar production. This project was undertaken to identify genes potentially involved in mediating nectary form and function in Brassica rapa
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